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Development of analytical methods for arsenic speciation Pergantis, Spiridon 1994

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DEVELOPMENT OF ANALYTICAL METHODS FOR ARSENICSPECIATIONbySPIRIDON PERGANTISB.Sc., University of loannina, Greece, 1988A THESIS SUBMI’fliiD IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFDOCTOR OF PHILOSOPHYinTHE FACULTY OF GRADUATE STUDIESDEPARTMENT OF CHEMISTRYWe accept this thesis as conformingto the required standardTHE UNIVERSITY OF BRITISH COLUMBIAAPRIL 1994© Spiridon Pergantis, 1994In presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.__________________Department of C—keMThe University of British ColumbiaVancouver, CanadaDate ES,, IS4.DE-6 (2/88)ABSTRACTMetal speciation has become increasingly important in making risk assessments ofmetal toxicities. No longer is the knowledge of the total elemental content adequate for sucha purpose, it is now essential to know the chemical form and concentration of all species ofthe 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 inenvironmental 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. Thespectra of the arsenicals exhibit molecular ions as well as characteristic fragment ions.To extend the usefulness of low resolution DCI-MS, an analytical method wasdeveloped that permitted the use of mass deficient reference standards for calibrationpurposes 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 forsimultaneous detection of the mass spectrum of perfluorokerosene (calibration substance)adequate for calibration purposes, and the spectrum of the analyte which contains molecularweight information. For positive ion accurate mass measurements of higher masses (up tom/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 compoundspresent in Mytilus californianus. This was accomplished by first extracting the arsenicalsfrom the mussel flesh with methanol, and then isolating and purifying the compounds bymeans of conventional chromatography. Subsequently the mass spectrometric techniquesdescribed above were used to analyze the purified materials and provide spectra suitable for11the 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 aslittle as 0.3 ng of arsenic or 4 pmole of the arsenical under investigation. This featuresuggests that the method could be used for the identification of minor arsenic components inenvironmental samples. Additional advantages of the technique are its capability of providingmolecular ions as well as fragment ions for a variety of arsenicals, and allowing for a certaindegree of control in compound fragmentation, by adjusting the N2 laser power. Quantitativeanalysis by MALDI-TOF-MS is still considered a highly unreliable procedure since manyvariables associated with sample preparation and analytical procedure can seriously affectthe results.Hydride generation - gas chromatography - mass spectrometry (HG-GC-MS),developed in this work, was used to provide conclusive evidence of -CD3 incorporationfrom L-methionine-methyl-d3into arsenic compounds produced from arsenate by alga cellcultures. These findings strongly support the notion that the oxidation-reduction pathwayinvolving carbonium ions, as originally suggested by Challenger for the alkylation of arsenicby microorganisms, applies to marine unicellular alga and probably other marine organisms.Micro - liquid chromatography (LC) columns (0.32 mm inner diameter) fabricated andpacked in house can be conveniently coupled on-line to a variety of mass spectrometricsystems, mainly because of the extremely low flow rates they require. It was shown that a99 % reduction in the volume of solvent waste is achieved by switching from conventionalto micro-LC, and a 87.5 % reduction by switching from microbore to micro-LC. Comparedto previous work, lower detection limits for arsenic compounds were also obtained whenmicro-LC columns were employed in conjunction with ultraviolet (UV) and electrothermalatomic absorption (ETAA) detectors.HIThe micro-LC columns are able to efficiently separate arsenicals that are used asanimal feed additives as well as their potential metabolites. The separation efficiencyachieved on this type of column was shown to be similar to those achieved on microbore (1mm inner diameter) and conventional (4.5 mm inner diameter) high performance liquidchromatography (}{PLC) columns.Simplex optimization was used to efficiently delineate the optimum experimentalconditions to be used for the electrothermal atomic absorption spectrometric analysis ofarsenic in a standard reference material of marine origin. Four experimental variables, wereconsidered: ashing temperature, atomization temperature, modifier concentration, andatomization ramping time. This combination of methods and materials provides a powerfulmeans of rapidly optimizing the experimental conditions used for the analysis of arsenic in awide variety of samples of environmental origin. Excellent recoveries of arsenic wereobtained when using the optimum electrothermal atomic absorption spectrometry conditionsto analyze standard solutions of arsenobetaine, arsenocholine and tetramethylarsoniumiodide. This procedure also allowed for the accurate determination of arsenic in Californianmussels collected from a variety of locations along the B.C. coast, which was found to rangefrom 9 to 16Finally, in Chapter 6 it was demonstrated that methylarsonic acid (MAsA) is a likelyprecursor to AsB in the marine environment. Mytilus calfornianus exposed for 9 days to aseawater system containing[3H]-methylarsonic acid, was found to contain [3HJ-.methylarsonic acid along with Hj-arsenobetaine and two unknown3H-labeled compoundsin the tissue parts of this mussel. A linear increase with time in the specific activity present inthe flesh of Mytilus calfomianus was also observed indicating uptake of the labelledcompound or its metabolites. The highest specific activity was found in the visceral mass andthe gills of the mussel.‘VTABLE OF CONTENTSABSTRACT iiTABLE OF CONTENTS VLIST OF TABLES xiLIST OFFIGURES xiiLIST OF ABBREVIATIONS xviiiACKNOWLEDGMENTS xxiiCHAPTER 1GENERAL INTRODUCTION 11.1 Arsenic Compounds Present in the Environment 21.1.1 Organoarsenic Compounds Present in Marine Organisms 21.1.2 Arsenic Biomethylation 41.2 Determination of Arsenic Species Present in Environmental Samples 61.2.1 Methods used for the Identification of Arsenic Compounds 71.2.2 Trace Speciation of Arsenic 71.2.2.1 Hydride Generation 71.2.2.2 High Performance Liquid Chromatography 81.3 Objective and Overview of Thesis 8CHAPTER 2DEVELOPMENT OF MASS SPECTROMETRIC METHODS FOR ARSENICSPECIATION 112.1 INTRODUCTION 112.1.1 Scope and Rationale of Work 112.1.2 Mass Spectrometry 142.1.2.1 Desorption Chemical Ionization 152.1.2.2 Matrix Assisted Laser Desorption Ionization 162.2 EXPERIMENTAL 172.2.1 Instrumentation 172.2.1.1 Low and High Resolution DCI - MS Instrumentation 172.2.1.2 MALDI-TOF-MS Instrumentation 17V2.2.1.3 Hydride Generation - Gas Chromatography - MSInstrumentation 172.2.2 Reagents 192.2.2.1 Reagents used for High Resolution - DCI - MS 192.2.2.2 Reagents used for HG-GC- MS 202.2.3 Procedures 202.2.3.1 Low Resolution DCI-MS Procedures 202.2.3.2 High Resolution DCI-MS Procedures 202.2.3.3 MALDI-TOF-MS Procedures 222.2.3.4 HG-GC-MS Procedures 222.2 RESULTS AND DISCUSSION 232.2.1 DCI - MS of Arsenic Compounds of Environmental Interest 232.2.2 Accurate Mass Measurements in DCI - MS 322.2.2.1 PFK as an Internal Reference Standard for AccurateMass Measurements in Ammonia DCI-MS 332.2.2.2 Fomblin (18/8) as an Internal Reference Standard forAccurate Mass Measurements in Ammonia DCI-MS 362.2.3 Analysis of Arsenic Compounds by using MALDI-TOF-MS 412.2.4 Structural Characterization of Arsines by using HG-GC-MS 502.4 SUMMARY 57CHAPTER 3OPTIMIZATION OF ATOMIC ABSORPTION SPECTROMETRIC METHODSFOR THE DETERMINATION OF ARSENIC IN BIOLOGICAL SAMPLES OFMARINE ORIGIN 593.1 INTRODUCTION 593.1.1 Scope and Rationale of Work 593.1.2 Atomic Absorption Spectrometry 603.1.2.1 Electrothermal Atomic Absorption Spectrometry andthe Determination of Arsenic 613.1.2.2 Hydride Generation Atomic Absorption Spectrometryfor the Determination of Arsenic 623.1.3 Digestion Procedures for the Preparation of Biological Samplesfor Arsenic Determination 62vi3.1.3.1 The use of Microwave Energy for the Digestion ofBiological Materials 643.1.4 Simplex Optimization 643.2 EXPERIMENTAL 673.2.1 Instrumentation 673.2.2 Reagents 683.2.3 Sample Preparation 693.2.3.1 Microwave Digestions Prior to ETAAS Analysis 693.2.3.2 Wet Digestion Procedure Prior to HGAAS Analysis 693.2.4 Procedure for the Simplex Optimization of ETAASExperimental Conditions 703.3 RESULTS AND DISCUSSION 723.3.1 Simplex Optimization of Conditions for the Determination ofArsenic in Environmental Samples by using ETAAS 723.3.2 Determination of Arsenic in Environmental Samples by usingHGAAS 783.3.2.1 Wet Digestion HGAA Determination of Arsenic 793.3.2.2 Microwave Digestion HGAA Determination ofArsenic 803.3.3 Comparison of Methods used for the Determination of Arsenicin Environmental Samples 823.3.4 Arsenic Concentrations in Californian Mussels Collected fromthe B.C. Coast 843.4 SUMMARY 87CHAPTER 4DEVELOPMENT OF HPLC AND MS METHODS FOR THE SEPARATIONAND DETERMINATION OF ARSENIC ANIMAL FEED ADDiTIVES ANDTHEIR METABOLITES 884.1 INTRODUCTION 884.1.1 Scope and Rationale of Work 884.1.2 Arsenicals used as Animal Feed Additives 904.1.3 Micro - HPLC used for the Separation of Arsenical Animal FeedAdditives 924.1.4 Interfacing Liquid Chromatography to Mass Spectrometry 94vu4.2 EXPERIMENTAL .964.2.1 Instrumentation 964.2.1.1 HPLC pumps 964.2.1.2 Injectors 974.2.1.3 HPLC Columns 974.2.1.4 HPLC Detectors 974.2.1.5 LC-MS Interfaces 984.2.2 Chemicals 994.2.3 Procedures 1014.2.3.1 Fabrication and Packing of Micro HPLC Columns 1014.3 RESULTS - DISCUSSION 1034.3.1 Separation and Determination of Arylarsenicals by using HPLCThermospray MS 1044.3.2 Separation of Arylarsenicals on a Microbore C18 HPLCColumn 1094.3.3 Separation of Arylarsenicals on Micro C18 HPLC Columns 1094.3.3.1 Effects of Packing Procedure and Particle Size on theQuality of Micro-LC Columns 1124.3.3.2 Effect of Column Length on the ElutionCharacteristics of the Arylarsenicals 1124.3.3.3 Effect of Temperature on the Elution Characteristics ofthe Arylarsenicals 1134.3.4 Detectors used for Microscale HPLC 1134.3.4.1 ETAAS used as a Detector for Microscale-HPLC 1144.3.4.2 Liquid Secondary Ion Mass Spectrometry 1164.3.4.3 Direct Liquid Introduction - Mass Spectrometry 1204.4 SUMMARY 122CHAPTER 5ARSENIC SPECIATION IN MYTILUS CALIFORNIANUS MUSSELS 1235.1 INTRODUCTION 1235.1.1 Scope and Rationale of Work 1255.2 EXPERIMENTAL 1265.2.1 Instrumentation 126vu’5.2.2 Chemicals 1275.2.3 Procedures 1275.2.3.1 Extraction of Organoarsenic Compounds from Mussels 1275.2.3.2 Purification Procedures for Arsenic CompoundsPresent in Mussel Extracts 1285.2.3.3 Isolation of Arsenic Compounds in the NH4OFraction 1295.2.3.4 Isolation of Arsenic Compounds in the HC1 Fraction 1305.2.3.5 Detennination of Arsenic in Mussel Shells 1315.3 RESULTS AND DISCUSSION 1315.3.1 Purification and Isolation of Arsenic Compounds Extractedfrom M. califomianus 1315.3.2 Identification of an Arsenic Compound Present in theAmmonium Hydroxide Fractions 1355.3.3 Identification of an Arsenic Compound in the Hydrochloric AcidFractions 1385.3.4 Attempts to Identify an Arsenic Compound in the WaterFractions 1405.3.5 Arsenic Speciation in Mussel Shells 1405.4 SUMMARY 142CHAPTER 6BIOTRANSFORMANTION OF[3H]-METHYLARSONIC ACID IN A STATICSEAWATER SYSTEM CONTAINING MYTILUS CALIFORNIANUS 1436.1 INTRODUCTION 1436.1.1 Scope and Rationale of Work 1436.1.2 Methylarsonic Acid in the Marine Environment 1446.1.3 Arsenic, accumulation and biotransformation experiments 1456.2 EXPERIMENTAL 1476.2.1 Instrumentation 1476.2.1.1 Liquid Scintillation Counting 1476.2.1.2 High Performance Liquid Chromatography 1476.2.1.3 Atomic Absorption Spectrometry 1476.2.2 Chemicals 1486.2.3 Collection and Storage of Mytilus californianus 148Ix6.2.4 Procedures for the Speciation of3H-Labeled CompoundsExtracted From Mytilus californianus 1496.2.5 Determination of Total 3H In Mussel Tissue and Shells 1506.3 RESULTS AND DISCUSSION 1526.3.1 Speciation of the3H-Labeled Compounds Extracted From M.californianus 1526.3.1.1 Identification of[3H]-MAsA and[3H]-AsB in musselextracts 1546.3.1.2 Attempts to identify Peaks #1 and #2 1606.3.2 Determination of Total 3H in Mussel Tissue and Shells 1606.4 SUMMARY 162CHAPTER 7SUMMARY 164REFERENCES 168APPENDIX A Thermospray mass spectra of arylarsenicals 183APPENDIX B LSIMS mass spectra of arylarsenicals 186APPENDIX C DLI mass spectra of test standards using heated capillary interface.. 189xLIST OF TABLESPAGE1.1 Some of the major arsenic compounds present in environmental and biologicalsystem 32.1 DCI filament currents at which arsenic compounds were desorbed 252.2 Positive ion accurate mass measurements of arsenic and other compounds in DCImode, using PFK or Fomblin 18/8 as internal reference standards 352.3 HG-GC-MS experimental parameters 523.1 Most common digestion methods used for the preparation of biological samples formetal analyses 633.2 Operating conditions for the Hydride Generation Atomic Absorption System 683.3 Range and precision of experimental variables examined 713.4 Simplex experiments and ETAAS response 743.5 Furnace heating program 763.6 Arsenic concentration found in NEST oyster tissue 763.7 Arsenic concentration of solutions containing organoarsenicals 773.8 Recovery of arsenic from DOLT-i and DORM-i 813.9 Concentration of arsenic in Californian mussels 853.10 Arsenic disthbution in Californian mussel tissue parts 864.1 Classification of Microscale HPLC 934.2 Summary of micro-column length vs resolution (R) obtained for arsenicals 1125.1 Weight and arsenic contents of extracted soft tissue, methanol extract, and residue 1326.1 Summary of chromatographic experiments 158XILIST OF FIGURESPAGE1.1 Mechanism for the biomethylation of arsenic, proposed by Challenger. Theintermediates in { } are unknown as monomeric species. They can be isolatedas (CH3AsO) and [(CH3)2AsJOin the laboratory, but not from anybiological system 51.2 Structure of S-adenosylmethionine (SAM) 52.1 Desorption probe equipped with a tungsten filament wire, 0.08 mm diameter,0.45 mm loop diameter, 8 loops 182.2 Negretti SS-needle valve (Model 145.207.2P2PK.IXE) system used to formspecific gas mixtures 182.3 Hydride generation - gas chromatography - mass spectrometry experimentalsetup. Dotted lines represent gas lines 192.4 Loading positions of sample and internal reference substance (Fomblin) on aKratos DCI probe filament 212.5 DCI mass spectrum of dimethylarsinic acid (0.2 big), ammonia reagent 242.6 DCI mass spectrum of arsenobetaine (0.2 pg), ammonia reagent 242.7 DCI mass spectrum of tetramethylarsonium iodide (0.2 tg), ammonia reagent 262.8 DCI mass spectrum of arsenocholine (0.2 .tg), ammonia reagent 262.9 DCI mass spectrum of trimethylarsine oxide (0.2 pig), ammonia reagent 272.10 DCI mass spectrum of disodium methylarsonate (0.2 .tg), ammonia reagent 272.11 DCI mass spectrum of sodium arsenate (0.2 rig), ammonia reagent 292.12 DCI mass spectrum of sodium arsenite (0.2 jig), ammonia reagent 292.13 DCI mass spectrum of o-arsanilic acid (0.2 jig), ammonia reagent 302.14 DCI mass spectrum of cL-toluenearsonic acid (0.2 jig), ammonia reagent 302.15 DCI mass spectrum of triphenylarsine (0.2 jig), ammonia reagent 31XII2.16 PFK mass spectrum obtained under mixed gas CI conditions. The ammonia tomethane ratio was monitored at m/z 18, and 17, 29 respectively. The relativeintensities and peak areas of these ions are presented above 342.17 Positive ion DCI mass spectrum of Fomblin using i-C4H12 as the reagent gas.The three major ion series observed are labelled as A, B, and C 372.18 Structural assignment of Fomblin fragment ions (A=69+n166; B=135+n166,and C=185+n166), obtained under positive ion DCI conditions, using iC4H12 as reagent gas 382.19 Positive ion DCI mass spectrum of Fomblin using NH3 as the reagent gas. Thethree major ion series observed are labelled as A’, B’, and C’ 382.20 Positive ion DCI mass spectrum of Fomblin using CH4 as the reagent gas 392.21 MALDI-TOF mass spectrum of arsenobetaine, DHB used as matrix(*:matrix ions) 442.22 MALDI-TOF mass spectrum of arsenobetaine, no matrix used 442.23 Effect of laser power (%) on the intensity of ions observed in MALDI-TOFmass spectrum of arsenobetaine, m/z 179 is the protonated molecular ion, m/z135 is the ion formed after loss of CO2.Each data point represents theaverage ion intensities 452.24 MALDI-TOF mass spectrum of tetramethylarsonium iodide, DHB used asmatrix. (*: matrix ions, DHB) 472.25 MALDI-TOF mass spectrum of arsenocholine, DHB used as matrix.(*: matrix ions, DHB) 472.26 MALDI-TOF mass spectrum of disodium methylarsonate, DHB used asmatrix. (*: matrix ions, DHB) 482.27 MALDI-TOF mass spectrum of p-arsanilic acid, DHB used as matrix.(*: matrix ions, DHB) 482.28 MALDI-TOF-MS calibration graph obtained from the analysis of MB (m/z179 corresponds to [M+H]). The molecular ion of DHB (mlz 154) was usedas an internal standard 502.29 Mass spectra of arsine, methylarsine, and dimethylarsine are presented. Asolution containing standard arsenate, methylarsonic acid, and dimethylarsinicacid (50 ng of arsenic for each compound) was used to generate the arsines 53XIII2.30 A: Mass spectrum obtained from culture medium inoculated with Lmethionine-methyl-d and arsenate, average of 100 scans. B: Mass spectrumobtained from culture media inoculated only with arsenate, average of 100scans 552.31 Full scan and Selected Ion Chromatograms (SIC) obtained from the analysis ofculture media inoculated with both L-methionine-methyl-d3 and arsenate. a.Full scan mlz 74-115 b. SIC of m/z 112 c. SIC of m/z 109 d. SIC of m/z 93 563.1 A simplex for two variables. A, B: simplex moves in a direction opposite theworst point; C: simplex “climbs’1 response surface and reaches optimum 663.2 Schematic diagram of hydride generation assembly 673.3 Wet Digestion apparatus. A: Teflon® cylindrical plugs; B: Teflon® diffusionfunnel; C: Teflon® stopper with capillary; D: 250 mL round-bottomed flask,containing sample and acid mixture. Adapted from the design described byBajo et al.17° 703.4 The absorbances obtained from 33 optimization experiments plotted as afunction of the four experimental parameters studied. Dotted lines indicate theestimated highest response level at this value of experimental parameter,irrespective of the values of the other three parameters 753.5 Typical calibration plot obtained by ETAAS. Dotted line indicates 95%confidence limits; r: correlation coefficient; b: slope; a: intercept; S: standarddeviation 823.6 Typical calibration graph obtained by HGAA. Dotted line indicates 95%confidence limits; r: correlation coefficient; b: slope; a: intercept; S: standarddeviation 834.1 Structures of arsenic compounds used as animal feed additives 894.2 Continuous flow LSIMS probe and Cs ion beam 984.3 DLI nebulizers; A. diaphragm nebulizer with desolvation chamber, and B.heated capillary nebulizer 1004.4 Fabrication of the micro-HPLC column 1024.5 Schematic diagram of the apparatus used for packing micro-HPLC columns 1034.6 Methods used for the separation and identification of animal feed arsenicals 104xiv4.7 A: Three dimensional graph showing the variation of resolution between 3-NHPAA and 4-NPAA when the methanol content of the mobile phase and theflow rate are varied. B: UV Q=254 nm)-chromatogram of arylarsenicals (22.5ng As of each compound) 1064.8 Thermospray mass spectrum of p-arsanilic acid. Temperature settings: Probe at129 °C, vaporizer at 185 °C, source block at 223 °C, and jet at 120 °C 1084.9 Calibration plot obtained by using Thermospray-MS for p-arsanilic acid and 4-nitrophenyl arsonic acid. The error bars represent the signal range obtainedfrom three injections of the analyte 1084.10 UV (=254 nm)-chromatogram of arylarsenicals (2.5 ng As of eachcompound); Microbore C18 column, 80 pL.miir1flow rate, 85% water(0.1% TFA) and 15% methanol 1114.11 UV (A=254 nm)-chromatogram of arylarsenicals (0.8 ng As of eachcompound); micro-HPLC column, 10 pL.min4flow rate, 85% water (0.1%TFA) and 15% methanol. A: Compounds separated: p-ASA, 3-NHPAA, and4-NPAA B: 4-hydroxyphenylarsonic acid added to analyte mixture of A 1114.12 Effect of temperature on the arylarsenical retention times 1134.13 ETAAS.-chromatograms of arsenicals, separated on a: A. conventional-LCcolumn, 500 pL fractions collected, B. microbore-LC column, 40 j.tL fractionscollected, C. micro-LC column, 5 i.iL fractions collected 1154.14 LSIMS mass spectra of 3-NHPAA; A. Negative ion detection, and B. Positiveion detection (*: NBA matrix ions) 1184.15 Background subtracted CF-LSIMS mass spectrum of p-ASA; mobile phase:89% water (0.1% TFA), 10% methanol, and 1% glycerol (Glyc); flow rate; 5pL.min1 1194.16 DLI-MS of 3-NHPAA, mobile phase consisted of 90% water (0.1%TFA),and 10 % methanol, 20 iL.min4flow rate, negative ion detection 1214.17 DLI-MS of 4-NPAA, mobile phase consisted of 90% water (0.1 %TFA), and10 % methanol, 20 iL.min’ flow rate, negative ion detection 1215.1 Proposed mechanism for the biosynthesis of arsenosugars by marine algae 1245.2 Proposed mechanisms for the biosynthesis of arsenobetaine 1265.3 Structure of an arsenic containing phospholipid present in brown alga 133xv5.4 Sephadex LH-20 - ETAA chromatogram; mobile phase: water; 7 mL fractionscollected; 20 i.iL of each injected into the ETAA spectrometer; each fractionanalyzed in triplicate 1345.5 Dowex 50Wx8(H+)- ETAA chromatogram; mobile phase: 120 mL water, 120mL 5% ammonium hydroxide, 50 mL water and 200 mL 2M HC1; 7 mLfractions were collected; 20 pL of each fraction was injected into the ETAAspectrometer; each fraction analyzed 1345.6 HPLC-ETAAS chromatogram of: A. Synthetic arsenic standards: AsB, As(llI),and DMAsA; 20 pL samples containing 500 ng of each arsenical wereinjected onto the column, B. arsenic containing material collected off thestrong cation exchange column 1365.7 DCI (ammonia reagent gas) mass spectrum of arsenic containing material,collected off a strong cation exchange resin in the ammonium hydroxidefractions 1375.8 DCI (ammonia reagent gas) mass spectrum of arsenic containing material,collected off a strong cation exchange resin in the hydrochloric acid fractions... 1395.9 HG-GC-AAS chromatogram of arsenic containing species, originating fromCalifornian mussel shells 1416.1 Structures of glycerylphosphorylarsenocholine (6. 1A), and phosphatidylarsenocholine (6. 1B). Both of these compounds were present in yellow-eyemullet following oral administration of arsenocholine 1466.2 Seawater tanks used for mussel storage and exposure experiment: A: 200 Lseawater tank with continually flowing seawater, used for mussel storage; B:15 L experiment tank containing mussels and[3H]-MAsA; *: aeration linesfor bothAandB 1486.3 Oxygen Combustion Flask (3L); A. platinum basket, B. paper sample wrapper,C. pressure relief in the form of a rubber balloon 1516.4 Sephadex LH-20 liquid scintillation chromatogram; water mobile phase; 9.5 mLfractions collected; 0.5 mL of each fraction mixed with 5 mL scintillate andcounted 1526.5 Dowex 50Wx8 (Hj liquid scintillation chromatogram; mobile phase 200 mLwater, 200 mL 5% ammonium hydroxide, 40 mL water and 150 mL 2M HC1;9.5 mL fractions collected; 0.5 mL of each fraction mixed with 5 mLscintillate and counted 153xvi6.6 HPLC conditions for A, B, C: Waters Protein Pak DEAE column; mobile phase5 mM sodium acetate, pH adjusted to 4 with acetic acid; flow rate 1 ml.min;fractions collected every 1 mm;A. HPLC-ETAA chromatogram of standards a: arsenobetaine (250 ng As),b:dimethylarsinic acid (500 ng As) , c: methylarsonic acid (500 ng As).B. HPLC-liquid scintifiation chromatogram of Peak #3; 1 mL fraction mixedwith 5 mL scintillate and counted.C. HPLC-liquid scintillation chromatogram of Peak #4; 1 mL fraction mixedwith 5 mL scintillate and counted 1556.7 HPLC conditions for A and B: Waters Protein Pak DEAE column; mobilephase 5 mM ammonium acetate, pH 6.8; flow rate 1 mLmin4.A. HPLC-ETAA chromatogram of standards a: arsenobetaine (250 ng As),b:dimethylarsinic acid (250 ng As); 0.5 mL fractions collected every 0.5 mm.B. HPLC-liquid scintillation chromatogram of Peak #4; 1 mL fraction mixedwith 5 mL scintillate and counted 1566.8 HPLC conditions for A, B, C: Bondclone 10 C18 reversed phase column;mobile phase water-methanol 95:5; ion-pair reagent 5 mMtetrabutylammonium nitrate; flow rate 1 mLmin4.A. HPLC-ETAA chromatogram of standards a: arsenobetaine, b:dimethylarsinic acid, C: methylarsonic acid; fractions collected every 30 sec.B. HPLC-liquid scintillation chromatogram of Peak #3; 1 mL fractioncollected and mixed with 5 mL scintillate and counted.C. HPLC-liquid scintillation chromatogram of Peak #4; 1 mL fractioncollected and mixed with 5 mL scintillate and counted 1576.9 Distribution of 3H activity within mussel parts, sampled on day 9 162xviiLIST OF ABBREVIATIONSAA atomic absorptionAAS atomic absorption spectrometryAES atomic emission spectroscopyAs(ffl) arsenite and/or arsenious acidAs(V) arsenate and/or arsenic acidAsB arsenobetaineAsC arsenocholineBC British ColumbiaCF continuous flowCI chemical ionizationcollisional induced dissociationCMS composite modified simplexDa daltonDCI desorption chemical ionizationDHB 2,5-dihydroxybenzoic acidDLI direct liquid introductionDMAsA dimethylarsinic acid and/or dimethylarsinateDMAsE dimethylarsinylethanolEl electron ionizationES electrosprayET electrothermalETAAS electrothermal atomic absorption spectrometryFAAS flame atomic absorption spectrometryFAB fast atom bombardmentFD field desorptionxv”FID flame ionization detectiong gramGC gas chromatographyGF graphite furnaceGlyc glycerolGPAC glycerylarsenocholineGPC gel-permeation chromatographyh hour(s)HCL hollow cathode lampHG hydride generationHGAAS hydride generation atomic absorption spectrometryHPLC high performance liquid chromatographyICP inductively coupled plasmaID inner diameterkg kilogramL literLOD limit of detectionLSIMS liquid secondary ion mass spectrometrymm minuteMALDI matrix assisted laser desorption ionizationMAsA methylarsonic acid and/or methylarsonateMBI moving belt interfaceMDA minimum detectable amountmm millimetermM millimolarMS mass spectrometry3-NHPAA 3-nitrophenyl-4-hydroxyphenylarsonic acidNIST National Institute of Standards and TechnologyNMR nuclear magnetic resonance4-NPAA 4-nitrophenylarsonic acidNRCC National Research Council of CanadaOD outer diameterp-ASA p-arsanilic acidp-UPAA p-ureidophenylarsonic acidPEG polyethylene glycolPFK perfluorokeroseneppb parts per billionppm parts per millionR resolutionRSD relative standard deviations secondSAM S-adenosylmethionineSIC selected ion chromatogramSIM selected ion monitoringSRM standard reference materialtR retention timeTDFC thermal desorption filament currentTFA trifluoroacetic acidTIC total ion chromatogramTLC thin layer chromatographyTMAs + tetramethylarsonium ionTMAsO trimethylarsine oxidexxTOF time of flightTSP thermosprayUV ultravioletwavelengthmicroliterxxiACKNOWLEDGMENTSI would like to express my deepest and most sincere gratitude to my researchsupervisors, Dr. W. R. Cullen and Dr. G. K. Eigendorf, for introducing me to this excitingresearch and sharing their knowledge and enthusiasm for it. Their guidance, support andencouragement during the course of my studies and research at the UBC are sincerelyappreciated.I would also like to thank Dr. A. P. Wade for introducing me to experimental design,in particular simplex optimization, and helping me apply it to my research project.My gratitude is extended to the technical staff of the department and the staff of themechanical workshop, electronic workshop, and mass spectrometry laboratory for theirinvaluable assistance on many occasions. I especially would like to thank Lina L. Madilao,Chris A. Emond, and Marshall Lapawa for their valuable suggestions and assistance inperforming many of the mass spectrometric experiments, as well as for the friendlyenvironment they created while I worked in the mass spectrometry laboratory.My greatest thanks go to my parents, brother and sister for their continued belief in meand for their support, which by no means was weakened by the geographical distance putbetween us.Finally, I would like to express my special thanks to my wife Alexandra for her love,patience, and encouragement during my studies, as well as to acknowledge the sacrifices shehas made by following me in pursuit of my Ph.D. degree.xxilCHAPTER 1GENERAL INTRODUCTIONArsenic occurs ubiquitously in the atmosphere, hydrosphere, pedosphere, lithosphere,and biosphere of the earth.’ The arsenic content of the earth’s crust is quoted to range from1.5 to 3 mg.Kg1 on average, however, this may vary depending on geographicallocations.”2 Arsenic is found in many types of mineral deposits, particularly thosecontaining sulfides and sulfosalts. The principal arsenic containing minerals are: realgar(As4S), orpiment (As2S3), arsenopyrite (FeAsS), and arsenides of copper, iron, cobalt,and lead.3Arsenic is mobilized into the environment mainly by natural processes such asweathering, volcanic, and biological activity. The environment also receives arsenic as aresult of anthropogenic activities such as smelting and burning of fossil fuels, industrial use,and mining activities.’ 4’5 The ratio of natural to anthropogenic atmospheric inputs hasbeen estimated to be 60:40.6Arsenic is well known for the toxic properties of some of its compounds. Arsenictrioxide (white arsenic, tasteless and colourless powder) was the favoured homicidalpoison7 through the Middle ages and until the nineteenth century. During the first WorldWar, Lewisite gas (C12AsCH=CHC1), a highly vesicant organoarsenical compound, wasused with severe effects. However, arsenic has also been used for medicinal purposes: asearly as the fourth century BC a paste of realgar was used as a treatment for ulcers. Duringthe early 1900s, arsenic compounds were found to be effective in the treatment of syphilisand other diseases.4’7Today, there is little use of arsenicals in human medicine.Industrial use of arsenic is, however, widespread.5Arsenic trioxide (As203)which isthe usual commercial form of arsenic, is used at the level of 50,000-53,000 tons per yearworldwide. Arsenicals are being used as wood preservatives, desiccants and herbicides in1cotton production. Even though various health and environmental protection organizationsand agencies have been trying to gradually restrict the use of arsenic, the production ofhigh-purity metallic arsenic for gallium arsenide semiconductors has been increasing. Inaddition, the use of arsenic compounds in animal feeds is still continued.Current scientific interest in regard to the arsenic biogeochemical cycle was initiatedat the beginning of the century when high levels of the element were found in marineorganisms. This interest is well justified, taking into account the fact that marineorganisms, via the food chain, are a major source of arsenic for human beings8 and thatthe various arsenic species exhibit different toxicities.1.1 Arsenic Compounds Present in the Environment1.1.1 Organoarsenic Compounds Present in Marine OrganismsDuring the 1920s it was established that marine organisms naturally contain highlevels of arsenic, relative to the levels present in seawater.9”°In 1925 Cox reported thatwithin 24 h of a person eating fish, arsenic was detected in the urine at levels previouslythought to be associated with chronic arsenic poisoning.11The first successful isolation and identification of an organoarsenical originatingfrom a marine animal was reported by Edmonds et at..12 This arsenical, present in the tailmuscle of the western rock lobster, was characterized by using X-ray analysis as beingarsenobetaine (AsB), Table 1.1. Since then, AsB has been confirmed to be the majorarsenical present in marine animals such as fish, crustaceans, molluscs, andechinoderms.L1320 Meanwhile, toxicological studies have established the non-toxicnature of MB.21’2 The second arsenical reported to be present in marine animals isarsenocholine (AsC), however conflicting reports concerning its presence have beenmade.2327 Other arsenic compounds identified in marine animals are2tetramethylarsonium ion (TMAs)28 and trimethylarsine oxide (TMAsO).29 The majorarsenic compounds of environmental interest are listed in Table 1.1.101112131415NameArseniteArsenateMethylarsonic acidDimethylarsinic acidTrimethylarsine oxideTetramethylarsonium ionArsenocholineArsenobetaineDimethylarsinylethanolArsenosugarsR(CH3)2As(O)-(CH3)2As(O)-(CH3)2As(O)-(CH3)2As(O)-(CH3)2As(O)-(CH3)As+-FormulaAs(OH)3H3AsO4CHAsO(OH)2(CH3)AsO(OH)(CH3)AsO(CH3)4As+(CHAs-t-CH2CO(CH3)As-i-CHCOO-(CH2As(O)CHCOx-OH-OH-OH-NH2-OH-OHAbbreviationAs(ffl)As(V)MAsADMAsATMAsOTMAs+AsCAsBDMAsEY-OH-OPO3HCH2CH(OH)CH-SO3H-SO3H-OSO3H-OSO3HTable 1.1 Some of the major arsenic compounds present in environmental and biologicalsystems.No.1234567893The ubiquity of dimethylarsinyiribosides (arsenosugars) in marine macroalgae hasbeen well established.3039 These compounds have also been found in a clam kidney(Tridacna maxima), probably derived from symbiotic unicellular green algae within theclam tissue.35 Most of the arsenic speciation studies concerning arsenosugars have beencarried out on Japanese edible seaweeds. The water soluble arsenicals extracted frombrown algae, Ecklonia radiata36’7 and Hizikia fusiforme37’8 were identified as thearsenosugars 10-14 (Table 1.1). The first tetraallcyl arsenic compound to be isolated from amarine algae is the trimethylarsonioriboside Recent work on the kidney of the giantclam Tridacna maxima has shown the presence of an arsenic containing nucleoside, inaddition to the arsenosugars.4°Attempts have been made to identify lipid-type arsenic (ether-soluble arsenic,chloroform-soluble arsenic) in marine algae. These compounds were identified asphospholipids containing a dimethylarsinyiribosyl moiety.41,421.1.2 Arsenic BiomethylationThe biological methylation of arsenic, has been observed to occur in a variety ofmicroorganisms, as well as in higher organisms such as plants, mice, monkeys and man.43Challenger was the first to investigate the mechanism of biomethylation in detail.’ In hispioneering work he identified as trimethylarsine (Me3As) the poisonous gas produced bymolds growing on wall paper coloured with arsenic containing pigments. The mechanismhe proposed for the methylation of arsenic (Figure 1.1) involves sequential reduction, andoxidative transfer of a methyl group from a CH3+ donor to arsenate.’45 Sadenosylmethionine (SAM) (Figure 1.2) is the likely source of the methyl group. Anumber of compounds such as thiols and dithiols including cysteine, glutathionine,dithiothreitol, and lipoic acid have been found to be capable of carrying out the reductionvia a two electron transfer.46’74H3AsO4 2& > H3AsO[CH3] CH3AsO(OH)2 2e{CH3As(O )2} [CH3] (CH3)2AsO(OH) 2& {(CH3)2As(OH)}[CH3f (CH3)AsO. 2& > (CH3)AsFigure 1.1 Mechanism for the biomethylation of arsenic, proposed by Challenger. Theintermediates in { } are unknown as monomeric species. They can be isolated as(CH3ASO)n and [(CH3)2As]0in the laboratory, but not from any biological system.Figure 1.2 Structure of S-adenosylmethionine (SAM).0$51.2 Determination of Arsenic Species Present in Environmental SamplesFor a complete understanding of the biogeochemical cycle and the toxicologicalsignificance of arsenic, it is necessary to know all the chemical forms of the element that areinvolved.Three general approaches have been employed for the speciation of environmentalarsenic compounds. The first approach involves the separation of the arsenic species from alarge quantity of sample. After the compounds are purified and isolated, their structures aredetermined by using mass spectrometric techniques, together with in some cases NMRspectroscopy, X-ray crystallography, and elemental analysis. These procedures allow forunequivocal identification, however they also demand large amounts of sample and muchtime and effort. In addition there is the possibility that isolation and work up procedures canproduce artifacts which do not exist in the original source.The second approach for the identification of arsenic species, involves the use ofefficient and mild separation techniques coupled to highly selective and sensitive detectionmethods. Such methods are suitable for both qualitative and quantitative analysis, but only ifappropriate arsenic standard compounds are available.485°However, compounds can bemis-identified if they happen to coelute.The third approach involves the use of similar chromatographic techniques coupledon-line to mass spectrometric systems which are capable of offering fairly high sensitivity,as well as providing a moderate degree of chemical information.61.2.1 Methods used for the Identification of Arsenic CompoundsNovel arsenic compounds have been discovered and identified in various marineplants and animals only after they had been completely isolated from their natural matrix.’320 Various types of chromatography have been used for this purpose: i.e. gel permeation,ion-exchange, thin layer, and high performance liquid chromatography. A variety ofstationary and mobile phases have been used in conjunction with these systems.X-ray diffraction analysis can provide complete structural identification of thearsenical, providing that a large amount of sample is available (usually about 1 mg) incrystalline form. NMR spectroscopy also provides much structural information, but for 13CNMR studies usually several tens of micrograms of sample are required. Mass spectrometry(MS) can provide structural information adequate for identification purposes with sub-microgram down to sub-nanogram amounts of material. The amount required is highlydependent on the MS technique employed. For complete identification, it is also necessaryto use high resolution MS.1.2.2 Trace Speciation of Arsenic1.2.2.1 Hydride GenerationA number of arsenic compounds can form hydrides upon treatment with sodiumborohydride under acidic conditions. Arsenite and arsenate both form AsH3, however,arsenite can be selectively determined by carefully adjusting the pH of the reduction.553Methylarsonic acid (MAsA) forms CH3As2,while dimethylarsinic acid (DMAsA) forms(CH3)As , and TMAsO forms (CH3)As, upon reduction with sodium borohydride.5456The hydride generation (HG) technique has been coupled with a variety of spectrometrictechniques, particularly atomic absorption spectrometry (HG-AAS), allowing forextremely low limits of detection for the arsines.576° Major improvements in thespeciation capabilities of the method were made when a gas chromatograph was7incorporated between the hydride generator and the detector.61’2 The use of a massspectrometric detector allowed for additional structural information regarding the arsinesproduced.63A major limitation associated with the technique is its inability to detect someenvironmentally important arsenicals such as AsB, TMAs+, AsC, and arsenosugars,because these compounds do not form hydrides upon treatment with sodium borohydride.However, the problem can be eliminated by using an appropriate digestion procedure priorto hydride generation. Recently on-line microwave digestion64 and on-line UVdecomposition65 procedures have been introduced to extend the usefulness of hydridegeneration methods.1.2.2.2 High Performance Liquid ChromatographyHPLC is a very efficient separation technique for arsenic compounds. Ionic arsenicspecies can be easily separated by using anion or cation exchange chromatography,6668as well as on reverse phase ion pair HPLC.687°Appropriate counter ions, such as thetetraalkylammonium cation or heptanesulfonate anion are added to the mobile phase. Gel-permeation HPLC has been used for the separation of arsenosugars.27’69The use of HPLC coupled to highly selective and sensitive detectors has lead tosignificant advances in element speciation research. The most common detectors used are:flame atomic absorption spectrometry (FAAS),71’2 electrothermal atomic absorptionspectrometry (ETAAS),70’34atomic emission spectrometry (AES), and plasma MS,particularly inductively coupled plasma mass spectrometry (ICP-MS).48501.3 OBJECTIVE AND OVERVIEW OF THESISThe main objective of this work was to develop analytical methods for the speciationof arsenic at trace levels. Applications of these methods were made on samples of8environmental interest, in order to advance our understanding of the arsenic cycle in theenvironment.Chapter 2 describes the development and evaluation of a variety of massspectrometric techniques such as Desorption Chemical Ionization (DCI) - MS, MatrixAssisted Laser Desorption Ionization (MALDI) -Time of Flight (TOF) - MS. and HG-GCMS, for use in the structural identification of arsenic compounds of environmental interest.The advantages of DCI-MS for analyzing this type of material were explored. Forconclusive identification of unknown arsenic compounds, a high resolution accurate massmeasurement DCI method for positive ions, was developed. A relatively new massspectrometric method, MALDI-TOF-MS was also evaluated for its potential to provideimproved sensitivity over other MS methods used for the analysis of arsenicals. Finally, aHG-GC system was interfaced to a mass spectrometer and was used to identify deuteriumlabeled arsenicals.Chapter 3 outlines the optimization of AAS methods, that are used for the accuratedetermination of arsenic in samples of marine origin. A Simplex optimization procedure wasused in conjunction with a standard reference material (SRM) of marine origin in order toefficiently delineate the optimum experimental conditions for the analysis of arsenic by usingETAAS. Four experimental variables were considered: ashing temperature, atomizationtemperature, modifier concentration, and atomization ramping time. Microwave dissolutiontechniques were also evaluated for their ability to digest marine samples containing arsenic.Chapter 4 describes the fabrication and packing of micro-HPLC columns and theirinterfacing to mass spectrometers. The primary concern of this work was to developanalytical micro methods capable of separating and determining organoarsenicals used asanimal feed additives. A number of analytical systems were evaluated in this study.9In Chapter 5, the speciation of arsenic in Californian mussels (Mytiluscalifornianus) is described. DCI-MS, both low and high resolution (described in Chapter2) was used to characterize arsenicals that were obtained from purified extracts of musseltissue.In Chapter 6, investigations concerning the biotransformation of an arseniccompound in the marine environment are described. This particular study is concernedwith the uptake and biotransformation of3H-labeled methylarsonic acid({3H]-MAsA) in astatic seawater system containing Californian mussels, Mytilus californianus.10CHAPTER 2DEVELOPMENT OF MASS SPECTROMETRIC METHODS FORARSENIC SPECIATION2.1 INTRODUCTION2.1.1 Scope and Rationale of WorkA brief account of arsenic compounds present in the marine and terrestrialenvironment has been given in Chapter One of this Thesis. A great amount of work has goneinto the development of analytical methods capable of identifjing these compounds. To alarge extent this work has been successful and has advanced our understanding of the arseniccycle in the environment.1J3However, these investigations need to be continued in orderto complete our understanding of the arsenic cycle, and also to assess toxicity on the basis ofthe arsenic species present rather than just on the total arsenic content. This is of particularinterest, because the toxicity of arsenic greatly depends on the form in which the element ispresent.Arsenic speciation continuous to be a challenge for the analytical chemist. The workpresented in this chapter contributes to the improvement of analytical techniques used forthe speciation of trace elements, and it is hoped that this effort will allow for furtheradvancements in arsenic speciation research. A variety of mass spectrometric techniques andprocedures were developed and evaluated for their use in the speciation of environmentalarsenic compounds.11Elemental speciation is defined as the identification and quantitation of all the physicochemical forms of an element present in the sample.76’7 These forms include gaseouscompounds, solid forms or phases and dissolved forms, depending on the nature of thesample. Also, the chemical form of an element refers to both the oxidation state, and thetype and number of substituents.Mass spectrometry (MS) has proven to be a very powerful tool for the identificationof arsenic compounds. The most informative mass spectra have been obtained by using fielddesorption (FD),237879 fast atom bombardment (FAB),29367880 atmospheric pressurechemical ionization,8’electrospray81’2and thermospray MS.74 The main advantage inutilizing these soft ionization techniques is the detection of the compound’s molecular ion+. . +M or, more usually, a simple molecular adduct [M+X] , where X= H, Na, K, etc. Theeven-electron protonated molecule [M+Hf is the most common ion containing theunfragmented molecule formed by these soft ionization techniques. However, thedisadvantage in many cases, is the absence of characteristic fragment ions, particularly atlower masses. These features are reversed in spectra obtained by using electron impact (El)MS, which yields mass spectra in which molecular ions M+ are rare and fragmentationextensive.48 In order to overcome these deficiencies other MS techniques, such asDesorption Chemical Ionization (DCI), Matrix Assisted Laser Desorption Ionization - Timeof Flight (MALDI-TOF), and Hydride Generation - Gas Chromatography - MassSpectrometry (HG-GC-MS) have been studied in this Thesis. These techniques provide bothmolecular and structurally important fragment ions and have improved sensitivities fordetection of analytes. Frequently the high detection limits of commonly used methods do notallow the identification of minor arsenic containing species found in environmentalsamples.23,29,36,788012DCI-MS, is a technique that is easily accessible in most laboratories and capable ofproviding both molecular ions as well as characteristic fragment ions, for the type ofcompounds investigated here.Furthermore a procedure has been developed which allows for high resolutionaccurate mass measurement of positive ions in DCI mode. 83 Data obtained by using thismethod allow for calculation of the elemental composition of the compounds investigated.Mass deficient reference standards [i.e. perfluorokerosene (PFK)J, normally used forcalibration purposes in mass spectrometry, do not provide adequate mass spectra underammonia chemical ionization conditions. In order to overcome this problem a procedureemploying a mixture of ammonia and methane as reagent gas has been developed. In highresolution accurate mass measurement experiments, this gas mixture allows for thesimultaneous detection of mass spectra of PFK adequate for calibration purposes, and ofspectra containing molecular weight information for the analyte. For positive ion accuratemass measurements of higher masses (up to m/z=2300), perfiuoropropylene oxide (Fomblin18/8) was used successfully as a reference standard under ammonia, methane, and iso-butaneDCI conditions.Extremely high sensitivity has been reported for MALDI-TOF-MS, and thisstimulated us to evaluate the technique for the analysis of organoarsenicals of environmentalinterest. A number of matrices, normally used in MALDI, were evaluated for their potentialto promote desorption and ionization of organoarsenicals. Other factors which affect thequality of the mass spectra were also investigated, these include: sample and matrixconcentration, as well as the effect of laser power on compound fragmentation. Attemptswere also made to aquire quantitative data.Finally, a HG-GC-MS system, capable of identifying arsenic hydrides, was developedand used to identify deuterium labelled arsenicals. This analytical technique was employed in13conjunction with a feeding experiment in order to establish the methylating agent responsiblefor arsenic methylation in biological systems.2.1.2 Mass SpectrometryMass spectrometry had its beginning in the work of J. 3. Thomson (19 10-20) andsubsequently of F. W. Aston (1920s), concerning the behaviour of ions in magneticfields.84’5 This enabled the determination of naturally occurring isotopic abundances ofmany elements. Since then, the evolution of MS has been extraordinary. MS is now beingused in a wide variety of analytical applications and is perhaps the most frequentlyencountered of all analytical techniques. This can be viewed as a consequence of thetechnique’s ability to provide both qualitative and quantitative information on inorganic andorganic analytes in complex mixtures, information concerning the structures of complexmolecules, the structure and composition of solid surfaces, and isotopic ratios of elements insamples. In addition, for many purposes MS has not only the required very wide dynamicrange but is also the most sensitive analytical method available today.The operating principle of the method is basically very simple. First, the samplecomponents are converted into ions (positive and/or negative), which are then separated onthe basis of their mass-to-charge ratios, and finally a suitable detector is used to convert thebeam of ions into an electrical or optical signal.The most critical aspect of MS is the ionization process. A great number of differentionization methods have been developed in order to accommodate the special requirementsset forward by the variety of different compound classes. In selecting the most appropriateionization technique for a particular sample, a number of factors must be taken intoconsideration: the physical and chemical properties of the sample, the ionization efficiency ofthe ion source, and the kind of information required from the analysis.142.1.2.1 Desorption Chemical IonizationDCI-MS was first introduced in 1973.86 The technique had an immediate impact onthe analysis of involatile biological substances, because it was more convenient and easy touse than field desorption. The technique has been used to provide structural information onhighly polar and nonvolatile compounds, such as oligopeptides,86 flavonoid glycosides,87fullerenes,88 oligosaccharides,89 alkaloids,90 macrocycles with molecular weights up to4400 Da,91 and various synthetic polymers (polystyrenes, polyethylene glycols,polysiloxanes, and polynorbornene),92as well as on a whole variety of other compounds.Some of the most advantageous features of this technique are the following: very fastanalysis time, good sensitivity, both molecular weight as well as structural information areobtained, and the method is easy to use and free of chemical noise. The main disadvantageof the technique has been the relatively short duration of the signal (2-5 sec), due to fastdesorption times. In the past this feature resulted in poor reproducibility of the massspectra.93 The introduction of fast scanning mass analyzers and data systems has allowedrapid data acquisition over wide mass ranges, thus solving this problem.During the DCI process, the sample is desorbed from a wire as a result of rapidheating. 93a,b This process takes place inside an ionization source in an atmosphere ofreagent gas. The gaseous sample molecules are ionized by collision with reagent gas ionsproduced by an electron beam. The CI source is constructed in order to maintain a reagentgas pressure of 1 torr inside the source and also maintain the analyzer region of thespectrometer at a pressure below I0 torr. A variety of different reagent gases have beenused in DCI.9495 In the positive ion mode the most widely used gases have been thosewhich yield Brönsted acids, some of which are: ammonia, methane, propane, and iso-butane.The major reaction mode of these gases is proton transfer to the analyte molecule. Innegative ion mode gases capable of forming BrOnsted bases are used successfully.152.1.2.2 Matrix Assisted Laser Desorption IonizationMALDI-TOF-MS was first introduced in 1988, by two independently workinggroups, those of Karas and Hillenkamp,96 and Tanaka and co-workers,97 and has sincebeen used with great success for the analysis of biopolymers,98 synthetic polymers,99peptides,100 oligonucleotides,10’and carbohydrates.102 The virtually unlimited massrange, and very high sensitivity are the two main features which have made this technique soattractive for various analytical applications.103 The latter feature, the high sensitivity,stimulated us to evaluate the technique for the analysis of organoarsenicals of environmentalinterest. Some of the disadvantages of the technique are the poor resolution achieved(reflecting the velocity of ejection of the ablated material), the unpredictability of the matrixselection for different compound types, and its poor reproducibility which makesquantitation marginal at best.The ionization process that is involved in MALDI is still not completely understood.The matrix used consists of an organic compound of low molecular weight (< 200 Da),capable of absorbing radiation in the 266-532 nm range. The analyte and matrix moleculesare intermixed in a crystallized lattice. Laser radiation (typically N2 laser emitting at 337 tim)is used to excite this lattice. The matrix is thought to absorb the energy and transfer it asprotonation to the analyte molecules. In some cases when the analyte contains certainchromophores capable of absorbing the laser radiation a matrix may not be required forobtaining ionization. The ions are formed in an electric field from which they are acceleratedinto a field-free drift region. This region is the TOF mass analyzer. Separation of the ions isbased on the fact that their velocities in this region depend on their mass-to-charge ratio.Thus small ions will reach the detector faster than larger ions of the same charge.103162.2 EXPERiMENTAL2.2.1 Instrumentation2.2.1.1 Low and High Resolution DCI - MS Instrumentation A quadrupole massspectrometer (Delsi/Nermag RiO-lOB) consisting of a desorption probe, an El/Cl source, aquadrupole mass filter, and a channeltron detector, was used for the low resolution DCIanalysis of the arsenic compounds. The desorption probe (Figure 2.1) carries a coiledtungsten filament wire, to which a heating current is supplied in order to thermally desorbthe sample after the probe has been inserted into the evacuated ion source chamber.High resolution (R=10,000, 10% valley definition) DCI measurements were carriedout on a double focusing mass spectrometer (Kratos Concept II H / Mach3 Data system). ANegretti stainless steel-Needle Valve system was constructed and used to supply gasmixtures of suitable ratios (Figure 2.2). A Kratos single reagent gas inlet system wasemployed to introduce the gases into the ion source and to control the ion source pressure.2.2.1.2 MALDI-TOF-MS InstrumentationTwo MALDI-TOF mass spectrometers were used for these experiments, one was aKompact MALDI (Kratos Analytical) and the other was a VG TOF (Fisons). Both wereequipped with N2 lasers emitting at 337 nm, 3 ns pulses. 100% laser power is equivalent to200 .iJ. The Kompact MALDI was capable of operating in the reflectron mode.2.2.1.3 Hydride Generation - Gas Chromatography - MS InstrumentationA quadrupole mass spectrometer (Delsi!Nermag Rb-bC) interfaced to a gaschromatograph (Varian, Vista 6000) was used in this study. A Porapak-PS (Waters,Milford, MA 01757) packed GC column was used for the arsine separations. Dataacquisition and processing were performed by using a PC based data system (Teknivent,Vector 2) interfaced to the mass spectrometer. The hydride generation apparatus consisted17of a peristaltic pump, a gas-liquid separator, a moisture trap, and a hydride trap. Theexperimental setup of the system is shown in Figure 2.3.Desozption ProbeThnsten FilamentFigure 2.1 Desorption probe equipped with a tungsten filament wire, 0.08 mm diameter,0.45 mm loop diameter, 8 loops.i-C4H’ >CH4NH3Kratos Single Reagent Gas InletFigure 2.2 Negretti SS-needle valvespecific gas mixtures.Negretti SS-Needle Valves(Model 145.207.2P2PK.1XE) system used to form18Peristaltic PumpGas/LiquidSeparatorFigure 2.3 Hydride generation- gas chromatography - mass spectrometry experimenta]setup. Dotted lines represent gas lines.2.2.2 ReagentsArsenobetaine (AsB),104 arsenocholine (AsC),105 tetramethylarsonium iodideçrr&s+I-)74and trimethylarsine oxide (TMAsO)106 were synthesized by using literaturemethods, all other chemicals were commercially available.Deionized water was used for all applications. Glass and plasticware were cleaned bysoaking them overnight in 2% Extran (BDH Inc.) solution, followed by a water rinse, asoak in dilute (2 M) hydrochloric acid and fmally a water rinse.2.2.2.1 Reagents used for High Resolution - DCI - MSThree reagent gases were used: i-butane, methane, and anhydrous ammonia (Linde,Union Carbide Canada Ltd.). Solutions containing various concentrations of Fomblin 18/8(Edwards, High Vacuum Canada Ltd. Oakville Ont.) were made up in PF-5060 (3M, St.Dry-Ice/Acetone Gas Chromatograph - Mass Spec.1. Arsine trap:Water TrapLiquid Nitrogen2. Releasing ArsinesWater bath19Paul, MN 55144-1000, USA). This solvent was preferred because it has a zero ozonedepleting potential.2.2.2.2 Reagents used for HG-GC- MSArsenic standards were freshly prepared by performing serial dilutions of stocksolutions (1000 ppm of elemental arsenic) of the following compounds: sodium arsenate,Na2HAsO4.7H0(MC&B, Norwood Ohio 45212); sodium arsenite, NaAsO2 (J.T. BakerChem. Co., Phillipsburg); disodium monomethylarsonate, CH3AsONa2.60(AlfaDivision, Danvers, MA 01923); dimethylarsinic acid, (CH3)2AsO(OH) (Sigma Chem. Co.,St. Louis, MO 63178). Solutions of 1 M HC1 and 2% (w/v) NaBH4were freshly preparedon a daily basis.2.2.3 Procedures2.2.3.1 Low Resolution DCI-MS ProceduresThe solid sample was dissolved in an appropriate solvent resulting in a 100 ppmarsenic solution. A syringe was used to apply 2 iL of the solution (200 ng of As) to theDCI filament. The solvent (usually MeOH) was allowed to evaporate prior to insertion ofthe probe into the ion source. After introduction of the ammonia reagent gas a heatingcurrent was applied to the desorption filament at a rate of 10 mA.s’. The sample wasthermally desorbed, and ionized by reagent gas ions that were formed via collision with anelectron beam.2.2.3.2 High Resolution DCI-MS ProceduresIn order to use PFK as a reference standard for accurate mass measurements undermixed ammonia-methane DCI conditions, it was first necessary to optimize the ratio ofammonia to methane. This was accomplished by adjusting the Negretti SS-Needle valve20system (Figure 2.2) to obtain the desired intensity of the reagent gas ions [CH5i’(m/z=17), [NH4] (mlz=18), and [C2H5] (m/z=29). Once this was done, PFK wasintroduced into the ion source via a heated inlet system. Subsequently the sample wasintroduced into the ion source by means of a DCI probe equipped with a filament. Thefilament current was ramped from 30 - 400 mA, in order to thermally desorb the sample.The filament current and heating rate were determined by the volatility and/or temperaturesensitivity of the analyte. This high resolution DCI-MS procedure allows for accurate massmeasurements of positive ions in peak matching, narrow scan or full scan mode.A slightly modified procedure was followed for accurate mass determinations ofpositive ions by using Fomblin as the internal reference standard. Only one reagent gas wasused and, because it is very difficult to introduce Fomblin into the ion source via a heatedinlet system, the sample and Fomblin were introduced simultaneously by using the DCIprobe. The sample solution was placed on the filament coil, while Fomblin, dissolved inPF-5060, was placed on the straight section of the filament (Figure 2.4).Figure 2.4 Loading positions of sample and internal reference substance (Fomblin) on aKratos DCI probe filament.Ceramic InsulatorSample212.2.3.3 MALDI-TOF-MS ProceduresSolutions (1.5 jiL) containing matrix [2,5-dihydroxybenzoic acid (DHB)] and analyte(typically 5 jig 10 ng), were applied on the sample slide (target) and allowed to air dryprior to their introduction into the mass spectrometer. After an initial “survey” scan acrossthe sample spot, the laser beam was focused on the solid phase matrix-analyte mixture in anarea were the signal to chemical noise ratio was optimized (often referred to as the “sweatspot”). The mass spectra reported in this thesis are average spectra obtained from 50successive laser shots.Internal calibrations were used in this work. Ions at mlz 23 ([Na]j and m/z 177({DHB+Na]j provided suitable reference ions for calibration.2.2.3.4 HG-GC-MS ProceduresFor the HG-GC-MS analysis, a semi-continuous method was developed based onmodifications of the procedures described by Kaise et alJ07 and Reimer.61 A peristalticpump was used to mix the sample (3 mL) with 1 M HC1 and 2% (wlv) NaBH4 solutions(Figure 2.3). Arsines were generated and swept through a gas-liquid separator by means of astream of helium gas and the evolved hydrogen gas, through a moisture trap (Teflon® U-tube, 30 cm length x 0.8 cm ID) which was cooled by a dry ice-acetone slurry, andsubsequently condensed in a hydride trap (Teflon® U-tube, 30 cm length x 0.4 cm ID)which was immersed in liquid nitrogen (-196°C). After the hydride generation reaction wascompleted the peristaltic pump was stopped and the hydride trap was warmed by using awater bath (70°C) to volatilize the arsines which were then carried to a Porapak-PS GCcolumn (80-100 mesh, 50 cm length x 0.4 cm ID, silanized with Silyl-8 [ChromatographicSpecialties] column conditioner as described by Reimer61). By using a gas chromatographwith a pre-set temperature program, the volatile arsines were separated and detected byusing a mass spectrometer.222.2 RESULTS AND DISCUSSION2.2.1 DCI - MS of Arsenic Compounds of Environmental InterestIn this work, the analysis of a number of arsenic compounds was studied by usingDCI-MS. The mass spectra obtained are presented and their features are discussed.The spectrum of DMAs (Figure 2.5) shows the protonated molecule at mlz 139, andalso a very characteristic fragmentation pattern. These fragment ions provide additionalstructural information and also allow for compound identification based on fourcharacteristic signals. This pattern is absent from spectra obtained by using thermospray74and atmospheric pressure chemical ionization,81 since both of these techniques providespectra which exhibit the even-electron protonated molecule [M+HfF of DMAs and nofragments at lower masses.Thermal desorption of DMAs occurred when the DCI filament current was at 170 mA(Thermal Desorption Filament Current, TDFC= 170 mA). The TDFC values of thecompounds studied are listed in Table 2.1.The spectrum of arsenobetaine (Figure 2.6) indicates a protonated molecule [M+H]+as well as a fragmentation pattern that again allows positive identification of this compound.Other methods such as FAB,80 electrospray,81 field desorption78’9 and thermospray74provide very informative spectra containing protonated molecular ions, and dimeric species,but fragmentation in some cases is observed to a much lesser extent. Published electronionization48 spectra do not show any molecular ion information.Tetramethylarsonium iodide exhibits a base peak at m/z 135, corresponding to thetetramethylarsonium ion (Figure 2.7), also present are fragment ions that are similar to thoseobtained for AsB.23-__________________________________________Dimethylarsinic acid r58 <(2M..OH.014Hsc_rto243 259OH25..< 91 186.,76 96 118 136 158 178198 218 238 258 278298 MfZFigure 2.5 DCI mass spectrum of dimethylarsinic acid (0.2 pg), ammonia reagent.135+H3[cH3As C—Ai-CH20075, 92 3Arsenobetaine56/Z5?179185I’[As]40 60 88 168 126 140 i6O 188 280 228 248 • 268 MIZFigure 2.6 DCI mass spectrum of arsenobetaine (0.2 pg), ammonia reagent.24Table 2.1 DCI filament currents at which arsenic compounds were desorbedArsenic Compound Thermal desorption filament current (TDFC)(mA)Arsenobetaine 210Trimethylarsine oxide 100Arsenocholine 200Tetramethylarsonium iodide 200Dimethylarsinic acid 170Disodium methylarsonate 250Sodium arsenite 270Disodium arsenate 320Triphenylarsine 0Arsenocholine exhibits an ion at m/z 147 corresponding to [M-H2O] plus othersignals that provide additional structural information (Figure 2.8). A low intensitymolecular ion peak is observed at m/z 165. Other methods result in molecular ion peaks ofa much higher intensity. In DCI-MS the presence of hydroxyl groups seem to mitigateagainst the observation of intense molecular ions, possibly due to reduced volatility.The base peak of the spectrum of trimethylarsine oxide corresponds to [M+HJ+ atm/z 137 (Figure 2.9). With the exception of triphenylarsine, the TDFC of 100 mA usedhere is considerably lower than the values recorded for the other organoarsenicals.Because of the relatively low volatility of the disodium methylarsonate (Na2-MMAs,MW= 184), a high TDFC (250 mA) was required in order to obtain its spectrum (Figure2.10). This resulted in an elevated temperature which is believed to be responsible for theformation of the arsenic dimers, trimers, and tetramers, present at masses m/z 150, 225, and300 respectively. Additional structural information can be obtained from the peaks at m/z137, 121, 106 and 92, as indicated in the spectrum.25i8øx [(CH3)As+ ]121 E(cH3)4As]135H3c—cu3Tetramethylarsonium Iodide58xF92185[Asj, I, •1 1h.j. • ?___.t L48 68 88 188 128 148 168 188 288 228 248 268 wzFigure 2.7 DCI mass spectrum of tetramethylarsonium iodide (0.2 gig), ammonia reagent.1884 44 II(CH3)As+H]+ x 2 —>CH3H3c_cH2cH2oHca3Arsenocholine[M-H2o]:58x 147133:[cH3As2]0Z5zi6558 78 98 118 138 158 178 198 218 236 258 278 wzFigure 2.8 DCI mass spectrum of arsenocholine (0.2 ag), ammonia reagent.2618B75x25:4486888 188 128 148 168 188 288 228 246 268 WLFigure 2.9 DCI mass spectrum of trimethylarsine oxide (0.2 rig), ammonia reagent.75z188z75y.58x25x137[M+H]fl3C—=cTrimethylarsine ox++Ocril1k -[As4]388I J.0II14.0— A,—ONa013Disodium methylarsonate296 3i8 338358 378 398 418 438 458 478 496 516 wzFigure 2.10 DCI mass spectrum of disodium methylarsonate (0.2 Mg), ammonia reagent.27Sodium arsenate and sodium arsenite both exhibit the same DCI mass spectra, Figures2.11 and 2.12 respectively, from which it is clear that DCI-MS is not able to distinguishbetween these two inorganic arsenic species. The high current of the DCI filament (TDFCarsenate=320 mA, TDFC-arsenite=270 mA) causes their pyrolysis and is probablyresponsible for formation of polymers. The presence of arsenate and arsenite might beindicated by the appearance of these polymeric species. Similar arsenic dimer, trimer, andtetramer formation has been reported to occur in AsH3 gas cells in the presence of heatedtungsten filaments.108o - Arsanilic (Figure 2.13) and p - arsanilic acid cannot be distinguished by thistechnique, since they produce identical spectra. The base peak for both appears at mlz 181[M-2H2Of, at mlz 94 we observe [H2NC65+ f, and at mlz 91 [AsOf. Arsenicdimers, trimers, and tetramers are observed at m/z 150, 225 and 300 respectively.- Toluenearsonic acid can also be identified by DCI-MS. A molecular ion at m/z 216(Figure 2.14) is observed, and in addition there are signals at mlz 198 due to [M-H2Of andat m/z 91 due to [C6H5CH2f’. The peaks appearing at m!z 257, 274, 306, 349, 365, and396 can be accounted for in the same manner as for the arsanilic acids.Triphenylarsine is desorbed from the DCI filament without applying a current. Theprotonated molecule [M+Hj+ forms the base peak in the spectrum (Figure 2.15), again thefragmentation pattern provides sufficient information to characterize this compound.A satisfactory full scan spectrum of these organoarsenicals is easily obtained with 100ng arsenic on the DCI filament. The ability of DCI-MS to detect organoarsenicals at suchlevels makes this technique eminently suitable for the investigation of environmentalsamples. To further reduce the detection levels one could utilize selected ion monitoring(SIM), which generally improves sensitivity by one to two orders of magnitude. We haveused the full scan DCI mode to identify AsB along with TMAs in mussel (Mytilus28californianus) extracts which had been purified by conventional liquid chromatographictechniques (Chapter 5, of this Thesis).LM1i,or 158 IA33191 225i’ r ih, i-’ii r . ..• 1.1•18 68 88 108 128 148 168 188 288 228 248 268(As4]Disodium ArsenateFigure 2.11 DCI mass spectrum of sodium arsenate (0.2 pg), ammonia reagent.188z75%58z25%188d7558%25‘887518875%58z25298 318 338 358 378 398 410 438 458 478 498 518 MJZI[AsT75[As2]158 [As3191. --18 68 88 188 128 148 168 188 288 228 248 268lAS4].Sodium Arsenite298 318338358378398418438458 478 490 518 wzFigure 2.12 DCI mass spectrum of sodium arsenite (0.2 pg), ammonia reagent.29[M-2H2O]8z75d’255a25Figure 2.13 DCI mass spectrum of o-arsanilic acid (0.2 pg), ammonia reagent.188w.75,25,[C6HsCH2J290 318 338 358 378 398 410 430 458 478 498 SIR WZFigure 2.14 DCI mass spectrum of -to1uenearsonic acid (0.2 pg), ammonia reagent.a-Toluenearsonic acid30Attempts were also made to analyze the above arsenicals in the negative ion DCImode. In most cases, fragments produced did not provide structural information about thecompound examined. AsB however exhibited a high intensity ion at mlz 177 ([M-H1 i.152(Ph2As.2H]51 1.69 TLPh2ASJ+-r .- •1 •18 68 88 188 128 148 168 188 288 228 248 268387[M+Hff.TriphenylarsineFigure 2.15 DCI mass spectrum of triphenylarsine (0.2 jig), ammonia reagent.188251887558>25298 318 336 358 378 398 418 438 458 478 498 516 wz312.2.2 Accurate Mass Measurements in DCI - MSAccurate mass measurements, of both fragment ions and of ions containing theunfragmented molecule (M+., [M+H]+, etc.), are of particular importance for theidentification of unknown arsenicals via the information thus provided on elementalcompositions of the ions. In suitable cases, generally at lower molecular masses, theelemental composition can thus be specified unambiguously, while in less favourable casesthe composition can be limited to a few possibilities. Thus, in order to increase the utility ofthe DCI mass measurements we investigated the possibility of making high resolutionaccurate mass measurements under DCI ammonia conditions. This procedure requires thepresence of an internal reference compound together with the analyte. However, theproblem associated with this requirement is that under ammonia DCI (or CI) conditionsnone of the normally used mass deficient reference standards, such as, PFK,heptacosafiuorotri-N-butylamine, or tris(perfluoroheptyl)-S-triazine, give adequate massspectra. Nevertheless, PFK has been used successfully with methane in chemical ionization(Cl)109 and DCI modes. PFK has also been used as an internal standard for accurate massmeasurements under alternating El and ammonia CI conditions.’1°In the latter procedurethe analyte is introduced into the ion source under ammonia CI conditions and several scansare acquired. The acquisition is suspended while the ammonia reagent gas is pumped out,PFK is then introduced into the ion source under El conditions.Substances other than mass deficient ones have also been used as calibrationreferences. Ballantine et. aL reported the use of mixtures of long-chain fatty acid methylesters, long-chain alcohol stearates, and carbowax 600 as standards for peak matching underammonia CI conditions. Because these compounds are not mass deficient, problems canarise in the measurement of unknown ions that have the same nominal mass as the referencestandard, unless high enough resolution is employed. Commercially available polyethyleneglycols (PEGs) and their mixtures have also been shown to serve as reference compounds32for full scan accurate mass calibration under ammonia CI conditions.’12These compoundsare also mass sufficient and precautions must be taken in order to avoid confusion.Furthennore, several mixtures must be prepared to cover a wide mass range.The lack of appropriate calibration substances for use in accurate mass measurementsof positive ions produced under ammonia DCI conditions has prompted us to investigate thesuitability of a number of substances for such a purpose. In this work we have demonstratedthe successful use of PFK as a reference standard under CI conditions when using a gasmixture of methane and ammonia. This procedure allows accurate mass measurements ofpositive ions in the full scan, narrow scan, and peak matching modes for a variety ofcompounds. Fomblin 18/8 was also established as a reference standard for DCI-MS incombination with either iso-butane, methane, or ammonia as the chemical reagent gas.Routine accurate mass measurement of compounds, with molecular weights up to 2300 Da,can be made in positive ion DCI mode.2.2.2.1 PFK as an Internal Reference Standard for Accurate Mass Measurements inAmmonia DCI-MSAs mentioned above, under CI conditions PFK yields adequate mass spectra withmethane as the ionization agent, but not with ammonia.109 Since it is desirable to analyzearsenic and other compounds under ammonia CI conditions, we investigated the possibilityof analyzing PFK in a gas mixture of ammonia and methane. By using defined mixtures ofthe two gases it is possible to simultaneously obtain mass spectra of PFK adequate forcalibration purposes, and satisfactory analyte spectra. The mass spectrum of PFK in amixture of NH3 and CH4, along with the required reagent gas ion ratios, is presented inFigure 2.16. Detailed mass assignments of PFK have been reported previously.109 The PFKspectrum obtained under mixed gas CI conditions is similar to the one obtained under Elconditions, with the exception that the relative intensities of the PFK ions above mlz 100 are33much higher under CI conditions. As expected, an increase in the ammonia to methane ratioresulted in suppression of the PFK ion abundances. Accurate mass measurements of arseniccompounds, along with analytes from other chemical classes, were made in order to validatethis procedure. Some of these results are presented in Table 2.2.[CHxl x2 [JJ]i ENH illI,.() fl.II I1.5 11.141 11.141 14.l1 14.44$ ,...41GAS PEAK PEAKIONS AREA TWTGHT[CH5] 0.68 0.86[NB II] 0.32 0.43[IJ,J 1.00 1.00293 493iiJiL[IbiiLLi1rr 1T 1T. 1 - I I j I I I100 800 800 400 800 800 700 800 M/ZFigure 2.16 PFK mass spectrum obtained under mixed gas CI conditions. The ammonia tomethane ratio was monitored at mlz 18, and 17, 29 respectively. The relative intensities andpeak areas of these ions are presented above.Reagent gas ion ratios100-50-0—.wz931I81iii‘43943J!jj’L [LII34Table 2.2 Positive ion accurate mass measurements of arsenic and other compounds in DCImode, using PFK or Fomblin 18/8 as internal reference standards.Compound / Reference I Ions Measured Theoretical Measured ErrorFormula Reagent gas mlz m/z ppmArsenobetaine/ PFK/NH3 [M+Hj 179.0053 179.0054 0.39C5H11O2As &CH4[(CH3)4Asf’ 135.0155 135.0157 1.48[(CH3)As+ ] 120.9998 120.9995 2.89[(CH3)2As] 104.9686 104.9690 4.29[CH3As2f 91.9607 91.9604 3.59[Asf 74.9216 74.9215 1.33Tetramethylarsonium PFK/NH3 [(CH3)4Asf 135.0155 135.0150 3.70ion / (CH3)4As & CH4[(CH3)As+Hj’ 120.9998 121.0003 3.72[(CH3)2Asf 104.9686 104.9690 4.29[CH3As2f 91.9607 91.9604 3.59[As] 74.9216 74.9212 5.34Monosaccharide / Fombim / [M+NH4]t 452.1768 452.177 0.43C18H2602 NH3Macrocycle I PFK / CH4 [M+H]t 489.1231 489.131 0.35C22H33O5BrSHemicarcerand / Fomblin / [M+H] 1430.7 197 1430.720 0.29C97508N3 i-C4H12Disaccharide / PFK /NH3 [M+NH4]t 598.2147 598.212 5.00C24H33F015 &CH4352.2.2.2 Fomblin (18/8) as an Internal Reference Standard for Accurate MassMeasurements in Ammonia DCI-MSSeveral groups have reported the use of Fomblin as a calibration substance fornegative ion Cl-MS.113-115 In this work we have investigated the use of Fomblin as acalibration substance for high resolution positive ion DCI-MS. Fomblin was analyzed in thepresence of three different reagent gases, i-C4H12 NH3, or CH4. The spectra obtainedwere evaluated for their suitability as calibration references.When i-C4H12 was used as CI reagent gas in the positive ion DCI mode, three majorion series were observed in the mass spectrum of Fomblin (Figure 2.17). The mlz values ofthese ion series can be expressed as A=69+n166, B135+n166, and C185+n166 (Figure2.18). The perfluoropropylene oxide (C3F60) units of Fomblin are believed to beresponsible for the 166 segment, while groups such as CF3 (m!z=69), C2F50 (m/z=135),and C3F70 (mIz=185) are believed to be the terminating groups of these oligomericFomblin fragments. The observed fragmentation pattern is in agreement with those reportedpreviously.113,116 The presence of these ion series throughout a wide mass range, frommlz= 100 to mIz=23 00, makes Fomblin (18/8) very suitable for use as an internal referencestandard for accurate mass measurements of positive ions under DCI mode, thussupplementing PFK at the high mass end (700 <m/z < 2300).When Fomblin was analyzed under NH3 CI conditions the resulting mass spectrumexhibited the series A’, B’, C’ (Figure 2.19), similar to those previously described ion seriesA, B, C but shifted upwards by 17 Da. Accurate mass measurements of the new spectrumrevealed the presence of NH3 adducts. The ion series A, B, and C were also present asminor components.Using CH4 as the reagent gas, the Fomblin fragmentation pattern differed from thepatterns discussed above (Figure 2.20). Under CH4 CI conditions a mass shift of the majorfragment ions was observed. This can be interpreted as being either a downward shift of the36)major ion series A by 22 Da or an upward shift of the ion series C by 28 Da. A downwardshift of ion series A by 22 Da may be attributed to the addition of an oxygen and the loss oftwo fluorine atoms, from the respective fragment ions (Scheme 2.1). An upward shift of ionseries C by 28 Da may be attributed to the capture of CO from the methane gas (Scheme2.2). The latter possibility was investigated by analyzing the CH4 reagent gas for thepresence of CO. It was found that only a minute amount of CO was present in the gasstream. Thus we believe that the CO in the gas stream is not responsible for the mass shift ofthe major Fomblin fragment ions under methane-Cl conditions, and that the proposedfragmentation pathway outlined in Scheme 2.1 is most favourable. However, it should bementioned that the major Fomblin ions observed under CH4 conditions are normally presentas minor signals in El mode as well as under CI conditions using NH3 or i-C4H12.Atoo.90-80-B‘“ C A1060-50c A40- 20151Z31A3O ian BA‘97c B1563B A20 4’3c a629C’ B C AI U a .Lk4800 1000 1200 1400 1600 1800 2000Figure 2.17 Positive ion DCI mass spectrum of Fomblin using i-C4H12 as the reagent gas.The three major ion series observed are labelled as A, B, and C.37ii m/zFF F1 235 FC5 899 As.. (I I CF6 1065 I i n 3PP7 12311 301 FFF FPFPC PCI I or Il5 965 B C2F5_O_(—6 1131 ii 3 ii7 1297 FF FP1 351 FFp FFpFC PC5 10156 1181 C3F7_O_(_C_C_O_)_ C ——I I7 13472F5 (FF PPFigure 2.18 Structural assignment of Fomblin fragment ions (A=69+n166;B=135+n166, and C=185+n166), obtained under positive ion DCI conditions, using ias reagent gas.ioo- A’A’ A’ 101290-A’80- A’ 141412487060 1510 A’1746A50 616 B’ C’ 1912982 1530 C’40 C’ C’ B’ is’sB’ C’ 1364 B’1862 fl’197620800 1000 1200 1400 1600 1800 2000Figure 2.19 Positive ion DCI mass spectrum of Fomblin using NH3 as the reagent gas.The three major ion series observed are labelled as A’, B’, and C’.3810090104380701209601111375761 977927 114330- 1541849 130920- I 1475 fbi10- fl__hi..i 1i__________________800 1000 1200 1400 1600 1800 M1Z 2000Figure 220 Positive ion DCI mass spectrum of Fomblin using CH4 as the reagent gas.Scheme 2.1FFF FFpFC PCI I -2F-Ec--c—o)-cpI 3 +0 —O<C3P4O)(-C—C—O---CPI I n-iFF PPIon Series Aii mz n m,z1 235 _p 1 2135 899 +0 5 8776 1065 6 10437 1231 7 120939Scheme 22FPp PFp\lf+Co PCc3r7—E--—?—o-)— —[c3p7—E-- —.—o-)— + co]PP pP nIon Series Cn m/z n nzfz1 351 +co5 1015 5 10436 1181 6 12.097 1347 7 1375Fomblin solutions of various concentrations were made up in PF-5060. This solvent isvolatile (b.p. 56 °C) and desorbs much earlier from the probe filament than Fomblin, thuseliminating an interference which could be caused by the solvent. Manufacturerspecifications list this solvent as containing C5-C18 perfluoro compounds. The massspectrum of the solvent showed ions up to m/z 328 (C6F14), which are of the samecomposition as those observed forPFK (m1z69, 100, 119, 131, 169, 219, 319).The procedure described above, using Fomblin 18/8 as reference, was evaluated foraccurate mass measurements of positive ions in DCI (CI) mode. Representative dataobtained from a number of compounds are included in Table 2.2.Data presented in this study indicate that mass deficient calibration referencecompounds such as PFK and Fomblin can be used successfully for high resolution accuratemass measurements in positive DCI (CI) mode. These compounds have a number ofattractive properties such as adequate volatility, mass deficiency, and compatibility with themost widely used CI gases or gas mixtures. The spectra consist of a series of reference ionscovering a wide mass range. Thus they are generally better reference materials for DCI-MSthan other compounds reported in the literature.” 1, 112, 117, 11840This investigation has shown that a gas mixture of NH3 and CH4 can be usedsuccessfully with PFK, while Fomblin can be used with either i-C4H12,NH3 or CH4 foraccurate mass measurements of positive ions in DCI (CI) mode. 832.2.3 Analysis of Arsenic Compounds by using MALDI-TOF-MSAs mentioned previously one of the objectives of our work was to investigate the useof the highly sensitive MALDI-TOF-MS technique for the analysis of low molecular weightarsenic compounds.The MALDI-TOF mass spectrum of AsB (Figure 2.21) shows a [M+Hf ion at m/z179, as well as [M+Naf and [M+Kf ions at mlz 201 and 217, respectively. The base peakof the spectrum appears at mlz 135 which corresponds to a loss of 44 amu (C02) from theprotonated AsB molecule. It is interesting to note that this four ion pattern is repeated forthe AsB dimer ion cluster. Proton, sodium, and potassium adducts of the dimer are presentat mlz 357, 379, and 395 respectively, while the protonated dimer after loss of CO2 is seenat mlz 313. Thus the pattern observed here, consisting of 8 ion peaks, serves as a uniquesignature for AsB. DHB was used as the matrix component.The approximate minimum detectable amount (MDA) of AsB was determined to be0.3 ng of arsenic or 4 pmole of compound. These values correspond to the total amount ofarsenic or compound respectively in the 1.5 .iL aliquot of solution deposited on the sampletarget. Because each mass spectrum is only generated from analyte desorbed from a verysmall area exposed to the laser beam, a large area of the solid phase matrix-analyte mixtureis not used and thus does not contribute to the signal obtained. Therefore the MDAsreported here could be improved by either reducing the solution droplet volume loaded ontothe target, effectively reducing the amount of analyte loaded, or by reducing the area ontowhich the solution droplet is deposited, thus allowing for the formation of a solid phasematrix-analyte mixture containing a much higher concentration of analyte.41Most publications reporting the use of mass spectrometry in organoarsenical structuraldeterminations have not stated the detection limits of the methods used or even the amountof sample needed to obtain suitable mass spectra.23293674788°(We are not referring toICP-MS, which is an element specific detector and only provides definite structuralinformation when used in conjunction with synthetic standards and chromatographictechniques). Therefore it is very difficult to compare the detection limits of the various massspectrometric methods used to analyze these compounds. One notable exception is the workof Siu and co-workers81’2who analyzed organoarsenicals by using atmospheric pressurechemical ionization and electrospray MS/MS. Initially they described the introduction of 1-100 ppm solutions of arsenic either continuously, or via flow injection. In SIM mode (mlz135) they reported a minimum detectable amount of 1 ng arsenic for AsB.81 In more recentwork where they used ion-exchange chromatography coupled to electrospray MS/MS theyreport the minimum detectable amount for AsB to be about 20 pg in the selected reactionmonitoring mode (m/z 179 [M+Hf/120 [M+H-C0-C3Ij.82As described above DCIMS can provide satisfactory full scan spectra of the organoarsenicals from 100 ng arsenic,when using ammonia as the reagent gas (Section 2.2.1).When comparing these various MS techniques, it is clear that MALDI-TOF-MS hashigh potential, especially since, as reported, it can provide mass spectra in the full spectrummode with very small amounts of analyte.96’8In order to investigate the effect that the matrix (DHB) has on the desorption andionization of AsB, mass spectra of the compound were also obtained in the absence of thematrix component. Figure 2.22 shows the mass spectrum of AsB obtained under suchexperimental conditions. A notable feature of this mass spectrum is the absence of anymolecular mass information; only the m/z 135 fragment ion is observed. A higher laserpower was required in order to obtain this spectrum, indicating that DuB plays a major role42in facilitating the desorption and ionization of AsB. DHB and its alkali impurities arerequired for protonation and alkali adduct ion formation.Laser photoionization TOF-MS has been previously used for the analysis of somearsenic compounds, mainly inorganic.119 The main feature of these spectra is theappearance of AsO+ from oxygen containing arsenic compounds; however, no detectionlimits or sample concentrations were reported.In the present investigation, using MB as a model, the effect of laser power onmolecular ion intensities as well as on fragment ion formation was studied. After an initialmaximum at 30% of full laser power the intensity of the protonated molecular ion of MBdecreases with increasing laser power. The intensity of the fragment ion at m/z 135 increasesinitially, but finally decreases at very high laser power (Figure 2.23). Both, the total ioncurrent and the ion at mlz 135 show an optimum at approximately 45% laser power. Fromthese results it is evident that, in general, the laser power can be selected so that molecularions, fragment ions or a combination of both can be observed.43fM+B-cOj+I1616161616767D161161161616161616l06CFigure 2.21 MALDI-TOF mass spectrum of arsenobetaine, DHB used as matrix (*: matrixions).l00161616ID7670IC416J16Pf9HJ+LM+NaV’I LM+K)1’4 2Jl 17.11 2I312M+H.O2Jt2M+HI135I(dflAs]lID IDI41WIiI¶H3H3C—-H2cOO-CH3a0 6III2 qI IDIlID 416 ZFigure 2.22 MALDI-TOF mass spectrum of arsenobetaine, no matrix used.44275025002250>1750150012501000750500250020 50 55 60Figure 2.23 Effect of laser power (%) [100 % laser power 250 jaJj on the intensity of ionsobserved in MALDI-TOF mass spectrum of arsenobetaine, mlz 179 is the protonatedmolecular ion, mJz 135 is the ion formed after loss of CO2.Each data point represents theaverage ion intensities from 50 laser shots. (TIC: total ion current, matrix: DHB)The tetramethylarsonium cation is another arsenic species which has been reported toexist in marine animals such as clams and mussels.28 The MALDI-TOF mass spectrum of asample of tetramethylarsonium iodide is presented in Figure 2.24. The base peak at m/z 135corresponds to the tetramethylarsonium species. An iodide adduct of thetetramethylarsonium dimer is observed at m/z 397.In comparing this spectrum with the data obtained from AsB it is clear that SIMdata82 are not sufficient to differentiate the various arsenicals.The presence of arsenocholine in the marine environment has been in dispute since itwas first reported in marine animals.2327 Most of the positive evidence is based onretention times obtained by using HPLC. The reported retention times match those ofsynthetic standards of arsenocholine, however no additional evidence was provided. TheMALDI-TOF mass spectrum of this compound is presented in Figure 2.25. A very strongmolecular ion (m/z 165) is observed along with some fragmentation (m/z 147, 135). Such25 30 35 40 45Laser Power (%)45spectral information would be invaluable in confirming the presence of AsC in environmentalsamples. This could be achieved by isolating HPLC fractions containing AsC and confirmingits presence by using MALDI-TOF-MS. Furthermore the development of dynamic MALDITOF should provide direct on-line MS capabilities. 1 19acDisodium methylarsonate [CH3AsO(ONa)2]was also analyzed by using MALDITOF-MS (Figure 2.26). The base peak at mlz 139 corresponds to protonatedmethylarsonate. In addition very intense sodium and potassium adducts are also observed atmlz 161 and 177, respectively. This ion pattern is also repeated for the methylarsonate dimerions.Another class of arsenicals worthy of investigating consists of the arylarsenicals thatare used as animal feed additives for swine and poultry. 120-12 1 Although these materialshave been used on large scale to promote production of these domestic animals for theconsumer market, very little analytical work has been carried out on these compounds. Oneof these compounds, p-arsanilic acid, was analyzed in this study (Figure 2.27). The maincharacteristics of the spectrum are the very intense protonated molecule as well as sodiumand potassium adducts of lower relative intensity. These compounds generally are verydifficult to analyze by MS methods, such as El or DCI, because they normally decompose orpyrolize on heatable MS probes. It should be noted that we have been able to successfullyanalyze these compounds by using dynamic - FAB, with MDAs at about 10 ng (Chapter 4,of this Thesis).4611SlaD‘aaaSSIsIsS.Figure 2.24 MALDI-TOF mass spectrum of tetramethylarsonium iodide, DHB used asmatrix. (*: matrix ions, DHB)IsIsaIsIsa,aDaIsSaI’IsS.I12z (cH)4As+IJ+a177IM] 165LM-B20]’135 147Is ‘ w’ Li..H3H3C_cH2cH2OHCa31 i,i ‘i1!_’ 7aoàá a _Figure 2.25 MALDI-TOF mass spectrum of arsenocholine, DHB used as matrix.(*: matrix ions, DHB)4W 47247139— LM-2Na+HJ 0H3L-0NaONaSULM-Na1— j (2M.4Na+H1+IM-2Na+K]• 177 277III 161• L2M-3Na]299S—257S L2M-4Na+KJ315$JiJk.• N I4•S lCD I SC SC SCFigure 2.26 MALDI-TOF mass spectrum of disodium methylarsonate, DHB used asmatrix. (*: matrix ions, DHB)SC 0SCD7’ NH.27.SSI”,EIDgIl4DIM+Ns1CD1 1M+KrKS /26137 240/CD*376•. I +1T, ICDI• .—,-.—I-• • SC SC aix SCFigure 2.27 MALDI-TOF mass spectrum of p-arsanilic acid, DHB used as matrix.(*: matrix ions, DHB)48Attempts were also made to analyze inorganic arsenic salts, (arsenite and arsenate) byusing MALDI-TOF-MS. The resulting mass spectra did not provide any meaningfulstructural information.Quantitative analysis by MALDI-TOF-MS is still considered a highly unreliableprocedure since many variables associated with sample preparation and analyticalprocedure can seriously affect the results. So far, only a small number of reportsconcerning quantitative analysis by using the technique have been made.122’3 In ourview the main problem is associated with inhomogeneity of the solid phase matrix-analytemixture. Thus, the resulting mass spectrum is highly dependent on the position of laserimpact. In our experience very poor precision is obtained not only between measurementsmade on different sample loadings, but also within the same sample deposition. Thisproblem can be resolved to a curtain degree by firing the laser beam at a large number ofdifferent spots on the matrix-analyte sample, either automatically or manually, andaveraging the resulting mass spectra. Another possible way of overcoming theinhomogeneity would be to recrystallize the solid phase matrix-analyte mixture on thetarget by using solvents, such as ethanol. Preliminary results from our investigations usingeither of these procedures did not produce analytically acceptable calibration curves for theorganoarsenicals. However, some improvement was observed when the matrix (DHB)molecular ion was used as an internal standard, Figure 2.28 shows a calibration graphobtained after following such a procedure.A recent report123 has claimed that the addition of nitrocellulose providesimprovement in sample-to-sample reproducibility of MALDI ion yields and also improvesthe precision for peptide quantitation. MS and optical microscopy results suggest that thenitrocellulose modifies the crystallization of matrix-analyte solution to allow even coverageover the sample surface.495.55.04.54.0I3.5i. 3.0.2 2.54-02.0C-J1.51.00.50.00Ln [amount of As(ng)]Figure 2.28 MALDI-TOF-MS calibration graph obtained from the analysis of MB (m/z179 corresponds to [M+H1). The molecular ion of DHB (mlz 154) was used as an internalstandard.All of the above arsenic compounds were analyzed in a variety of differentmatrices, including DHB, Sinapic (3,5-dimethoxy-4-hydroxycinnamic acid), HABA [2-(-4-hydroxyphenylazo)benzoic acidj, and gentisic acid. DHB provided the best results, both insensitivity as well as spectral information. The other matrices provided poor spectra.2.2.4 Structural Characterization of Arsines by using HG-GC-MSA need for HG-GC-MS analysis of arsines developed out of the work of H. Li.63”In his work Polyphysa peniculus was grown in artificial seawater in the presence ofarsenate and L-methionine-methyl-d3. The HG-GC-MS method described here was used todetermine if the -CD3 moiety from L-methionine-methyl-d is incorporated into arseniccompounds produced by the algae.1 2 3 450The principle of the method is based on the fact that inorganic and methylated arseniccompounds, i.e. As(OH)3 and (CH3)As(O)(OH)3 (n=O-3), can be reduced to theircorresponding arsines, (CH3)nAsH3n (n=O-3), by allowing them to react with sodiumborohydride. This reduction is a pH dependent process and pH control has been used tospeciate arsenate and arsenite.54’6”07125Under pH < 1 conditions, all hydride formingarsenicals can be reduced to the arsines. At pH > 4, arsenite but not arsenate forms arsine.The evolved arsines can be separated and detected by using gas chromatography - atomicabsorption spectrometry (GC-AA)27’9-31 and GC-MS.2961J7-129 The majority ofstudies, reporting the use of a mass spectrometer as a detector, used the selected-ion-monitoring (SIM) mode and demonstrated a capability for characterizing arsine,methylarsine, dimethylarsine and trimethylarsine. In a study involving the analysis ofarsenic in NaOH digested tissue extracts of shellfish, fish, crustaceans, and seaweeds, Kaiseet al.-°7 collected the arsines, generated by NaBH4 reduction, in a liquid-nitrogen cooledtrap that was coupled to a GC-MS system. The SIM mode gave detection limits of 0.3 ngarsenic. Earlier, Odanaka et al.’27 were able to quantify hydride forming arsenicals byusing a combination of a GC-MS system, an off-line hydride generator and a heptane coldtrap. The arsines were trapped in the cold heptane and the detection limits were between10-20 ng of arsenic. In almost all cases the most abundant ions of each arsine were used forthe SIM analysis, e.g. mlz 78 [AsH3Jand 76 [AsH] for arsine; m/z 92 [CH3As],90[CH3As] and 76 [AsHI’ for methylarsine; mlz 106 [(CH3)2AsHJ and 90 [CH3As] fordimethylarsine; m/z 120 [(CH3)AsJ, 105 [(CH3)2AsJ and 103 [(CH2)As] fortrimethylarsine.In the present study the mass spectrometer was scanned from m/z 74 to 115 at 1 scanper 0.1 s. Scans above m/z 115 were not made because there was no need to investigate fortrimethylarsine whose absence had been confirmed by using HG-GC-atomic absorptionspectrometry.’24The wide-scan monitoring mode was used instead of the SIM mode, for a51number of reasons. First of all only the wide-scan mode would allow for the observation ofany unsuspected fragment ions resulting from the deuterium labelled arsines. Thisprecaution was taken because the fragmentation patterns of deuterium labelled arsines apartfrom (CD3)As,13°were unknown. The other reason for acquiring data in the wide-scanmode was to establish the identity of any other volatile compounds resulting from the cellculture samples. Any such interfering ions could lead to erroneous results in the SIM mode.The HG-GC-MS experimental conditions used are all listed in Table 2.3. Theseconditions were established after optimizing the hydride generation efficiency, thechromatographic resolution, and the mass spectrometric sensitivity for the arsines ofinterest.The mass spectra of arsine, methylarsine and dimethylarsine obtained by using thewide-scan monitoring mode are presented in Figure 2.29. The present study was mainlyconcerned with the detection of dimethylarsine. By using the HG-GC-MS methodologydescribed above it was possible to detect down to 25 ng of arsenic. In the SIM mode thedetection limit could easily be improved by at least an order of magnitude.Table 2.3 HG-GC-MS experimental parametersHydride Generation Gas Chromatography Mass Spectrometer2% NaBH4 Initial temperature: 70°C Interface temperature: 110°C1 M HC1 Ramp rate: 30°C.min1 Ion source temperature: 140°C3 mL Sample Final temperature: 150°C for 2 mm Scan every 0.1 s for 4 mmHelium flow rate: 30 mL.min1 Scan range: m/z 74-11552eu90% [M-2] + AsH360%0%40%50%40%30%20%10%O%.170 75 90 95 90 95 100 105 110 115M!Z100*90% CH3AsH27670%40% [M]+50%40%30%20%10%[70 75 60 93 90 45 1QQ 105 110 115.M/ZFigure 2.29 Mass spectra of arsine, methylarsine, and dimethylarsine are presented. Asolution containing standard arsenate, methylarsonic acid, and dimethylarsinic acid (50 ngof arsenic for each compound) was used to generate the arsines.53I.100%90% (CH3)2AsH90%70%60%30%40%30%20% 7161:: t I I I I I70 75 60 IS 90 95MIZ100 205 110 115The data obtained lead to the conclusion that, when L-methionine-methyl-d3 is addedto the medium, the CD3 label is incorporated into the dimethylarsenic species to aconsiderable extent (in some cases up to 30 %). The mass spectra obtained from samplescontain ions at mlz 112 [(CD3)AsHf, m/z 109 [(CH3) DAsHf, mlz 94 [CD3AsHfand mlz 93 [CDAsJ+ (Figure 2.30A). These ions are absent from the mass spectra obtainedfrom the media containing no L-methionine-methyl-d (Figure 2.30B). Further evidence ofthe incorporation of the CD3 into the dimethylarsenic species is provided by the single ionchromatograms at m/z 93, 109, and 112, shown in Figure 2.31. It can be seen that peakretention times are identical with the retention time of dimethylarsine.The percentage of CD3 incorporation was determined by comparing the relative peakintensities of the molecular ions, m/z 106 [(CH3)2AsHf, 109 [(CH3) DA5HTF, and 112[(CD3)2AsH]t The relative standard deviation of the peak intensity of m/z 106 for 4determinations of standard dimethylarsinic acid was 6%. Also, the assumption that theionization efficiencies and thus the responses of the deuterated arsines are identical to theresponses of the undeuterated arsines may cause additional errors in the calculatedincorporation percentages. A previous study by Cullen et at’3° addressed this assumption,and concluded that the deuterated trimethylarsine shows slightly lower sensitivity under Elmass spectrometric conditions.54100%- I90% A80% [(CH3)2ASH]+70%[(CD3)(CH3)ASH] +60%I [(CD3)2AsHJ +— 50%1[CD3As]30% [44]+ I7520% I I191 110% 103 199 1120%- Hi. ii70 75 80 85 90 95 100 105 110 115MIZJ.uu.90% [CH3AsJ B80%70%60%50%40%30% [(CH3)2AsH)LAs] 120% 751110% I0%- I70 75 80 85 90 95 100 105 110 115MIZFigure 2.30 A: Mass spectrum obtained from culture medium inoculated with Lmethionine-methyl-d3 and arsenate, average of 100 scans. B: Mass spectrum obtainedfrom culture medium inoculated only with arsenate, average of 100 scans.55zzli—GO?Figure 2.31 Full scan and Selected Ion Chromatograms (SIC) obtained from the analysisof culture medium inoculated with both L-methionine-methyl-d3 and arsenate. a. Fullscan m/z 74-115 b. SIC of m/z 112 c. SIC of m/z 109 d. SIC of mlz 93.a0.—DOGCOOO0 0.2 0.’ 0.5 0.5 1.0 ‘.20..5 TRetention Time (s)l.2562.4 SUMMARYThe work which has been described in this chapter is mainly concerned with thedevelopment of mass spectrometric techniques for the analysis of arsenic compounds ofenvironmental interest. In particular, DCI MS was shown to produce mass spectra withcharacteristic ions and fragmentation patterns, thus providing abundant structuralinformation which could be used to analyze such compounds. This allows identificationbased on more ions than just the protonated molecular species.Accurate mass measurements can also be made by using this technique in conjunctionwith a high resolution mass spectrometer. Our investigations have shown that a gas mixtureofNH3 and CH4 can be used successfiully with PFK as reference, while Fomblin can be usedas reference with either i-C4H12, NH3 or CH4 as reagent gases in accurate massmeasurements of positive ions in DCI (CI) mode. This technique will be of particularimportance in identifying unknown arsenic species, as well as a wide variety of compoundswhich have been previously analyzed by using low resolution DCI-MS.We have also shown that MALDI-TOF-MS has great potential for the analysis oforganoarsenicals of environmental interest. Some of the main features of this technique areas follows:1. The method is capable of providing molecular ion as well as structural information for avariety of arsenic compounds. Molecular or quasi-molecular ions are observed in all theMALDI mass spectra presented in this work. In addition, in many cases structurallycharacteristic fragment ions and/or dimer formation along with sodium and potassiumadducts were observed. These features allow for the unambiguous identification of thearsenicals investigated so far.2. The flll spectrum detection limit of the method is extremely good compared to that ofother mass spectrometric methods reported. For instance, AsB, was detected at levels as lowas 0.3 ng of arsenic (4 pmole of compound).573. Only small volumes of sample are required for each analysis, <1.5 tL.4. The observed ion intensities are highly dependent on the N2 laser power. Higher powerresults in increased fragmentation coupled with a decrease in the intensity of the molecularions.5. Not all matrices normally used in MALDI-MS are suitable for the analysis: DHB is themost suitable matrix for the MALDI analysis of organoarsenicals.It is probable that MALDI-TOF-MS could be used, possibly with the same degree ofsuccess, for the analysis of other organometallics of environmental concern, particularlythose of Sn, Se, and Sb.The development of dynamic LC - MALDI-TOF-MS should increase the potential ofthe method to solve problems such as the ones referred to in this work.” 9a-cFinally a HG-GC-MS method was developed which provided conclusive evidence of -CD3 incorporation into arsenic compounds produced from arsenate by alga cell cultures.This evidence supports the hypothesis that 5-adenosylmethionine, or some closely relatedsuiphonium compound, is involved in the biological methylation of arsenic.58CHAPTER 3OPTIMIZATION OF ATOMIC ABSORPTION SPECTROMETRICMETHODS FOR THE DETERMINATION OF ARSENIC INBIOLOGICAL SAMPLES OF MARINE ORIGIN3.1 INTRODUCTION3.1.1 Scope and Rationale of WorkThe main objective of the work reported in this Chapter, is to optimize atomicabsorption spectrometric (AAS) methods in order to allow for accurate determinations oftotal arsenic in samples of marine origin. More specifically, we are concerned with thearsenic content of Mytilus calfornianus (Californian mussels) collected from the B.C.coast.One of the most commonly used AAS methods for the analysis of metals in varioussamples is Electrothermal atomic absorption spectrometry (ETAAS).131 Even thoughETAAS compares very well in terms of performance with most other analytical techniques,a number of interferences have been reported. These are especially pronounced whenanalyzing environmental samples. 1 32-134 In order to overcome these interferences, variousmodifications to parts of the atomic absorption spectrometer have been reported. Platformatomizers’35 and matrix modifiers’36have been used to overcome chemical interferences.Zeeman or deuterium background correction systems have been used to compensate formolecular absorption or light scattering.137 The performance characteristics of ETAAS areaffected by a wide range of instrumental components and fhrnace heating conditions.Because of the great variations in experimental parameters it is almost impossible to adopt a59set of experimental conditions established for the detennination of arsenic on one AAS system for the determination of the same element on a slightly modified AAS system. Evenconditions recommended for a broad category of samples (e.g. biological), may not beoptimum for a more specific sample type (e.g. oyster tissue), on the same spectrometer.It is therefore evident that an efficient optimization procedure must be adopted in orderto accomplish the accurate determination of arsenic in environmental samples. The majorityof reports to date have used one-factor-at-a-time optimization procedures.66’138 This has anumber of drawbacks; large numbers of experiments are required and the best conditionsmay be missed if important interactions exist between experimental parameters.In this study we have employed the Composite Modified Simplex (CMS)l39M°optimization method in conjunction with a Standard Reference Material (SRM) of marineorigin, in order to delineate the optimum experimental conditions for the analysis of arsenicby using ETAAS. Appropriate SRMs containing certified amounts of arsenic were selectedbased on the similarity of their matrix to the matrix of the marine samples of interest.3.1.2 Atomic Absorption SpectrometryAAS was first introduced by the Australian physicist Alan Walsh, in 1955.141 Thisanalytical method is based on the conversion of the sample components into gaseous atomswhich absorb radiation of wavelengths characteristic of their electronic transitions.Measurement of the amount of absorption allows a quantitative determination of the analyte.After some initial hesitation by scientists to utilize flame AAS for the determination ofmetals, the method soon saw extensive use in the 1970’s. Further development of the methodhas resulted in a highly reliable method which probably reached its development plateauseveral years ago.Meanwhile the introduction of other AAS methods have resulted in advances in thearea of trace metal determinations. L’vov first described Electrothermal (ET) or Graphite60Furnace (GF) AAS, three decades ago.142 This method offers great improvements in termsof sensitivity for the analysis of metals, and has since become the most widely usedanalytical method for such purposes.3.1.2.1 Electrothermal Atomic Absorption Spectrometry and the Determination ofArsenicIn ETAAS a very small volume (1-100 1.iL) is deposited onto the inner wall of agraphite tube, which is aligned in the path of light of an element-specific hollow cathodelamp (HCL). In some cases the solution is deposited on to a platform placed inside the tube.The graphite tube is then electrically heated in a number of steps,143 eventually resulting inthe vaporization of the sample and the formation of an atom cloud in the graphite tube.These atoms absorb light emitted from the HCL. The extremely high sensitivity of thetechnique for the analysis of metals can be attributed to a number of factors. First, theanalyte transport efficiency into the observation path is very high, close to 100%:techniques employing pneumatic nebulizers, such as flame and inductively coupled plasma(ICP), have very low analyte transport efficiencies, typically between 1-10%. Secondly,residence times of the analyte atoms in the observation path are about two orders ofmagnitude higher than those found in other sources. Thus, it is the high analyte densitiespresent in the observation path in ETAAS that are responsible for the high sensitivity andlow detection limits of the technique.ETAAS has been used widely for the determination of arsenic in biological,geological, marine and fresh water samples.1’31,44, 145 The United States EnvironmentalProtection Agency recommends ETAAS methods for the determination of a number oftrace elements, including arsenic.146 Low detection limits, good precision, simplicity of operation as well as minimum sample pretreatment are all features which have contributed tothe widespread use of the method.613.1.2.2 Hydride Generation Atomic Absorption Spectrometry for the Determinationof ArsenicMethods that employ hydride generation (HG) methodology coupled to atomicabsorption spectrometry have been used extensively for the determination of hydrideforming elements such as As, Bi, Se, and Sb,147 Ge,148 Te and Sn,’49 and Pb.’5° Suchanalyses are accomplished by first converting the metal or metalloid into its correspondinghydride, the hydride is then transferred to an atomizer where it is decomposed to the gas-phase metal (-bid) atoms. The atomization step takes place in the optical path of thespectrometer. The main advantage of this method is that it separates and preconcentratesthe analyte from the sample matrix. This procedures eliminates matrix interferences that arequite commonly encountered when analyzing samples by using other AAS methods, such asETAAS.HGAAS has been used extensively for the determination of arsenic in environmentalsamples. 53,151 Because of the nature of the technique only the hydride forming arsenicspecies will be detected. Since most samples of marine origin also contain non-hydrideforming arsenicals, these samples must be digested and converted to, e.g. arsenate, prior totheir analysis by using HGAAS. The digestion step is critical for the success of the analysis.If all the non-hydride forming arsenicals have not been converted into hydride formingspecies, low recoveries are obtained.643.1.3 Digestion Procedures for the Preparation of Biological Samples for ArsenicDeterminationBiological tissue samples are usually digested prior to their analysis, since mostquantitative analytical methods employing instruments allow for only liquid sampleintroduction. A variety of digestion methods have been used to convert a largely organicmatrix into a solution suitable for analysis.152-156 Acid digestions, as well as combustionmethods have been carried out in both open and closed vessels. A summary of the most62commonly used digestion techniques for decomposition of biological materials is given inTable 3.1.Table 3.1 Most common digestion methods used for the preparation of biological samplesfor metal analCommon Vessel Reagents Conditions Energy Advantages Disadvantagesname of type sourcetechniqueWet or Acid Open Acid Temperature Hot plate Specialized Large amountsDigestion mixtures, of acid equipment of reagents,oxidizing boiling points not required contaminationagent problemsMicrowave Closed Acid High Microwave Very fast, May not alwaysDigestion mixtures, pressures, energy typically accomplishoxidizing high 1-10 miii, completeagent temperatures on-line decomposition,digestions specializedequipmentOxygen or Closed Oxygen Flame Flame in Very fast, no Often difficult toSchoniger Combustion 02 environ- reagents dissolve ash,flask ment needed poor precisioncombustionDry Ashing Open Oxygen High Muffle Low Slow, possibleand temperatures Furnace amounts of analyte loss atashing (400-700 °C) reagents highaids required. temperatures,Can handle often difficult tolarge dissolve ash,amounts of contaminationsample problems‘sesIn order to select the appropriate digestion technique for the preparation of aparticular biological sample, a number of factors must be taken into consideration. Forexample, some analytical methods require that the metal under investigation be completelyremoved from its organic substituents. Other factors which must be taken into considerationare the following: the digestion vessel must not adsorb the metal of interest onto its walls or63allow the evaporation of the metal, also the digestion reagents must not interfere with theanalysis or contain impurities which may contaminate the sample under investigation.3.1.3.1 The use of Microwave Energy for the Digestion of Biological MaterialsMicrowave energy as a heating source for acid digestions was first introduced in1975.157 At that time these digestions were performed at atmospheric pressure.’58”9Because of the problems associated with open vessel digestions, researchers turned toclosed polycarbonate bottles160”62 and Teflon® PFA [(Perfluoro alkoxy) ethylene]digestion vessels.’62465 The main advantage of this procedure is that the digestion takesplace under high pressure, which allows the acid mixtures to reach temperatures above theboiling points of the acids. Microwave digestions have been used with great success forsample preparation of steels,’62 geological materials,163 and biological samples. 164l65The basic principle behind microwave heating is that microwave energy causesmolecular motion of ions and also dipole rotation, but does not cause changes in molecularstructure. Ionic conduction causes a flow of current which results in heat production, due toresistance to ion flow. Molecules with permanent or induced dipole moments are caused torotate in microwave electromagnetic fields, this results in rapid heating.3.1.4 Simplex OptimizationMost analytical procedures must be optimized prior to their use. A very popularmethod for such a purpose is the old one-factor-at-a-time method. This optimizationprocedure investigates the effects of several factors (experimental variables) on a response,by holding all but one factors constant. The responses obtained at the various levels of thisfactor are evaluated. Successive factors are then allowed to vary, while at the same timekeeping the previous factors at their optimum levels. However simple this procedure seems,there are a number of disadvantages associated with it. It is a time consuming and tedious64procedure, and most importantly it does not guarantee that an optimum will be reached,especially if there exists an interdependence of the optimum values of the chosen factors.In order to avoid these problems other more advanced statistical methods have beendeveloped. Simplex optimization, first introduced by Spendley et al.’66 in 1962 has beenshown to be quite successful in solving these problems. Deming and Morgan167 were thefirst to employ the procedure to optimize methods in analytical chemistry.To understand how simplex optimization works, let us consider a chemical system forwhich we want to optimize n variables (n=l,2,3,...). These variables (factors) can beconsidered as n orthogonal axes, with each experiment being represented by one point inthis n dimensional space. A simplex is defined as a geometric figure consisting of (n+1)vertices. So for a 2 variable chemical system, the simplex will be a triangle, for a 3 variablesystem it will be a tetrahedron, etc. So for n variables, n+1 experiments must be conductedin order to construct the first simplex.To better illustrate how the simplex optimization procedure functions, let us considera two variable system. The initial simplex will be a triangle, which is defined by the initialtrial points labelled 1,2, and 3, as shown in Figure 3. 1A. If we assume that the worstresponse was obtained at point 3, it would be logical to assume that a better response wouldbe obtained at point 4 (Fig. 3. 1A), which is a reflection of point 3 with respect to the linejoining 1 and 2. A new simplex is formed, consisting of points 1,2, and 4. By repeating thisprocedure the simplex “climbs” the response surface, and thus arrives at the optimumexperimental conditions (Fig. 3. lB and C). To increase the chances of reaching a globaloptimum, as opposed to a local one, the search can be repeated by starting off with differentinitial vertices.The optimization procedure described above is referred to as the fixed-step or basicsimplex method. Because of the limitations associated with this procedure, other moreadvanced simplex procedures, called modified simplex methods, were developed. These65methods allow for the acceleration of the simplex in favorable directions and deceleration inunfavourable directions. The expansion and contraction of the simplex results in fasteroptimization as the simplex “climbs” up the response surface and then contracts onto thetop.168 In the present work a modified version described by Betteridge et al. was used.’690Variable 1Figure 3.1 A simplex for two variables. A, B: simplex moves in a direction opposite theworst point; C: simplex “climbs” response surface and reaches optimum.A4F663.2 EXPERIMENTAL3.2.1 InstrumentationAn Atomic Absorption Spectrometer (Varian Techtron Model AA 1275) equippedwith an arsenic hollow cathode lamp (Spectra AA) operating at 8 mA, and a deuterium background corrector were used for the arsenic determinations. The 193.7 rim arsenic resonanceline was selected and used with a 1 nm bandwidth.For the ETAAS analysis the spectrometer was equipped with a GTA-95 (Varian)graphite furnace. Pyrolytically coated graphite partitioned tubes and argon purge gas werealso used.For the HGAAS analysis a continuous flow system, shown in Figure 3.2, wasemployed. The experimental conditions used are listed in Table 3.2.peristaltic pump________Figure 3.2 Schematic diagram of hydride generation assemblySampleAcidlight path____regulatorsgaslinesgas/liquid linesgas,‘liquid separator67Table 3.2 Operating conditions for the Hydride Generation Atomic Absorption SystemMaterialUptake tubes Sample: 2.80 mm I.D. PVCHC1: 2.28 mm I.D. VitonNaBH4: 2.29 mm ID. PVCUptake flow Sample: 7.5 mLminHC1: 2.0 mL.min1NaBH4: 4.OmL.min’Carrier gas N2: 100 mL.min1mixing coilN2: 25 mL.min1 gas-liquid separatorHC1 concentration 4 MNaBH4concentration 2 % (w/v) in 0.05% NaOH solutionMeasurement mode Run Mean3.2.2 ReagentsA 1000 ppm stock solution of arsenic, as arsenic trioxide, was used to preparearsenic standard solutions. A 1000 ppm palladium solution, the matrix modifier, was alsoprepared by dissolving the appropriate amount of Pd powder in the minimum amount ofaqua regia, followed by dilution with water containing 2% citric acid to the requiredvolume.Standard solutions containing 100 ppm of arsenic as arsenobetaine, arsenocholineand tetramethylarsonium iodide were also prepared. These compounds were synthesized byusing literature methods.74,104,105 Microelemental analysis and nuclear magnetic resonance spectroscopy were used to confirm their purity.Sample solutions containing microwave digested oyster tissue were used for theoptimization of the ETAA experimental conditions. The freeze dried oyster tissue, standardreference material 1566a, was obtained from the U.S. National Institute of Standards andTechnology (NIST).68Two other SRMs were also used in order to evaluate arsenic recoveries for theHGAAS method. Dogfish muscle reference material (DORM-i), and dogfish liver referencematerial (DOLT-i) were obtained from the National Research Council of Canada (NRCC).3.2.3 Sample Preparation3.2.3.1 Microwave Digestions Prior to ETAAS AnalysisA commercial microwave oven (Sharp Carousel II) was used to digest samplescontained in a Teflon® decomposition vessel (Parr Instrument Company, 45 mL).Samples of 150-400 mg each were weighed directly into the digestion vessel and 2 mLof concentrated nitric acid was added. The digestion vessel was assembled and placed in themicrowave oven. The microwave program consisted of one 90 s step at high power output(500 W).After cooling the digestion vessel the content was diluted to 50 mL with de-ionizedwater. Blanks and arsenic standards were also prepared by using 2 mL of nitric acid and thesame digestion and dilution procedure.3.2.3.2 Wet Digestion Procedure Prior to HGAAS AnalysisHomogenized mussel flesh or SRMs were freeze dried, 0.25-0.5 g of the sample wasput into a 250 mL round bottom flask, along with 3 mL 30% hydrogen peroxide, 3 mL 69%nitric acid, and 1 mL sulfuric acid. A specially designed apparatus (Figure 3.3) was used forthe digestion. This apparatus was originally designed and used for the digestion of biologicalsamples for Se, As, and Hg analysis. L70The digestion apparatus was placed in a heating mantle and heated for 3 h atapproximately 250 0C. The digestion was carried out in a fume hood. Finally the digestatewas transferred into a 100 mL volumetric flask.69Figure 3.3 Wet Digestion apparatus.A: Teflon® cylindrical plugs; B:Teflon® diffusion funnel; C: Teflon®stopper with capillary; D: 250 mLround-bottomed flask, containingsample and acid mixture. Adaptedfrom the design described by Bajo etal.’7°H 15mm-550 mm3.2.4 Procedure for the Simplex Optimization of ETAAS Experimental ConditionsThe simplex optimization was carried out as follows. Four variables were studied fortheir effect on arsenic absorbance; ashing temperature, atomization temperature, modifierconcentration, and atomization ramping time. The experimental variable names were enteredinto the microcomputer together with their ranges and the precision required for each70variable (Table 3.3). All the simplex caiculations were carried out by using the OPT1MA3computer program.171 This program was run on several PC/XT and PC/AT compatibleIBM microcomputers. The initial set of conditions was entered and the program thengenerated the four other sets needed to form the initial simplex and printed worksheets foreach experiment. After the completion of the experiments, the actual variable valuesused and the peak area absorbances were entered. The variable values used were kept asclose as possible to those suggested by the program.Table 3.3 Range and precision of experimental variables examinedVariable Lower limit Upper Limit PrecisionAshing temperature (°C) 600 1800 100Atomization temperature (°C) 1800 2700 100Modifier concentration (ppm) 25 500 25Ramp time (see) 0.5 8 0.5The program then calculated the next single set of conditions to be investigated andprinted another worksheet. This process was continued, the program giving one newexperiment each time. For each set of conditions three replicates were analyzed by usingETAAS. Between each one a blank injection was made to correct for possible lamp driftand to also assure that stable repeatable analytical results could be obtained.The optimum conditions obtained from this procedure were then used to analyzestandard arsenic solutions and quantify the arsenic present in the microwave digested oystertissue. These conditions were also used to analyze de-ionized water solutions containing arsenobetaine, arsenocholine and tetramethylarsonium iodide.713.3 RESULTS AND DISCUSSION3.3.1 Simplex Optimization of Conditions for the Determination of Arsenic inEnvironmental Samples by using ETAASVarious problems have been encountered when analyzing arsenic by using ETAAS,some of which are volatilization losses, interaction with the graphite tube, vapour phase interferences, and spectral interferences.172,173 Furthermore the analysis of arsenicespecially in samples of marine origin may pose additional problems. It is well documentedthat a large number of arsenic compounds are present in marine organisms.1 Therefore ifthe appropriate experimental conditions are not selected it is possible that each arsenicalmay behave in a different way during ETAAS analysis. This results in sensitivity variationsfor different arsenicals (due to incomplete detection) and therefore leads to results highlydependent on the arsenic species present. These species-dependent effects may be missed ifonly standard arsenic solutions of artificial origin are examined. Consequently it is necessaryto optimize the ETAAS conditions using ‘real” samples. SRMs which have a similar matrixto the environmental samples of interest are ideal for use in optimizing and validating themethod.NIST oyster tissue was used in this study. Table 3.4 shows the optimizationexperiments performed and the responses obtained. Twenty three experiments wererequired before establishing optimum conditions for the analysis of arsenic in oystertissue. A total of 33 experiments were performed before ending the simplex optimizationsearch. The recorded absorbance from each experiment as a fbnction of variable value isdisplayed in Figure 3.4.The optimization procedure very quickly predicted the optimum ramping time. Thiswas 0.5 sec (the minimum limit used in our optimization) and was reached after 13experiments. Instrumental limitations did not permit use of a shorter ramping time. These72results indicate that shorter atomization ramping times improve the arsenic absorbanceobtained in ETAAS. Longer ramping times probably allow for loss of arsenic attemperatures close to the atomization temperature.The optimum ashing temperature established in this study was 1500 °C. Thistemperature allows for the removal of matrix components which may otherwise act asinterferences for the analysis of arsenic. Higher temperatures result in arsenic loss duringthe ashing stage, while lower temperatures may result in incomplete removal of variousmatrix interferences.The optimum atomization temperature established was 2200 °C. We have shown thatthis temperature allows for complete atomization of all arsenic in the SRM analyzed.The optimum modifier concentration was 500 ppm (upper limit value). Modifier concentrations are not very critical when analyzing standard arsenic solutions, but areextremely critical when analyzing environmental samples. This is probably a consequenceof the sample’s matrix. In environmental samples higher concentrations of Pd are requiredbecause it interacts with various matrix components and may become unavailable to act as amatrix modifier for arsenic. However, use of higher concentration values for Pd results inhigher costs per analysis.73Table 3.4 Simplex experiments and ETAAS responseExpt. Cycle* Abs. [Pd] Ashing Atomization RampNo. (a.u.) (ppm) (°C) (°C) (sec)1 01 0.018 200 700 2000 42 0.019 200 1300 2000 43 0.015 200 1000 2400 44 0.010 200 1000 2100 75 0.016 500 1000 2100 56 1R 0.025 350 1200 2000 1.57 E 0.021 400 1300 2000 0.58 2R 0.000 500 1200 1800 1.59 C 0.026 300 1100 2200 1.510 3R 0.010 50 1200 2100 0.511 C 0.018 450 1000 2100 3.512 F 0.021 400 1100 2100 3.013 4R 0.042 450 1600 2200 0.514 E 0.031 500 1800 2400 0.515 F 0.038 400 1500 2200 0.516 5R 0.029 500 1600 2300 0.517 6R 0.030 450 1800 2300 0.518 7R 0.032 500 1800 2500 0.519 8R 0.037 500 1800 2300 0.520 9R 0.028 400 1800 2200 0.521 C 0.037 500 1600 2300 0.522 F 0.035 450 1700 2300 0.523 1OR 0.049 500 1500 2200 0.524 E 0.040 500 1400 2200 0.525 F 0.03 1 500 1600 2300 0.526 hR 0.026 500 1300 2000 0.527 C 0.030 500 1700 2400 0.528 12R 0.027 500 1300 2200 0.529 E 0.038 500 1700 2300 0.530 13R 0.045 500 1600 2200 0.531 14R 0.041 500 1300 2100 0.532 15R 0.040 500 1300 2100 0.533 E 0.038 500 1400 2100 0.5* I: Initial cycle, R:reflection, E: expansion, C:contraction, F:fit.Sample standard deviation of 10 blank determinations: 0.002 a.u.74005_______________________A 0.05,-“a,A0.04 IA 0.04-,A A AA0• A 0 / AI— / AL< 0.03 / f I < 0.03 Ao A I - A0 A&QQ2 0..• ‘0.02 AAA.0 A *00.01- : A 0.01 A A0.00 I 0.00 I I1600 1800 20D0 2200 2400 2600 1000 1500 2000Atomization Temp. (°c) Ashing Temperature ( C)0.05 Al 0.05A’0.04 0.04 ‘‘_VCq) ,, V0.03-a < 0.03o 2.o ,.‘a)0.02 A à.. 0.02 ,/V(I A 0 V.0 .0 “ V0.01 0.01 -.,‘ V0.00 A I I 0.00 I I0 1 2 3 4 5 6 7 8 0 100 200 300 400 500 600Romping Time (s) [Pd] (ppm)Figure 3.4 The absorbances obtained from 33 optimization experiments plotted as afunction of the four experimental parameters studied. Dotted lines indicate the estimatedhighest response level at this value of experimental parameter, irrespective of the valuesof the other three parameters.75The optimum furnace operating conditions along with the solution volumes used inthis study are given in Table 3.5.Table 3.5 Furnace heating programaStep No.: Temperature Time (s) ArgonPurpose (°C) Flow(mL.min 1)1: Drying 85 5.0 32: -II- 95 40 33: -II- 120 10 34: Ashing ramp to 1500 5.0 35: -II- 1500 1.0 36: -II- 1500 2.0 37: Atomization ramp to 2200 0 Abs.measured8: -II- 2200 2.0 0 Abs.measured9: Cleanup 2200 2.0 310: -II- ramp to 2600 0.5 3a: Sample and matrix modifier solutions (20 jiL of each ) injected onto ETAAS.Underlined parameters were optimized.To check if these conditions also resulted in optimum arsenic recovery, the normalcalibration method was used to determine arsenic in the NIST standard. The standardadditions method was not required. Three samples were microwave digested and analyzedfor arsenic, under the optimum ETAAS conditions. The arsenic recovery results obtainedfrom these experiments, in addition to the certified values of arsenic in the SRM, aredisplayed in Table 3.6. These results indicate that the experimental conditions allow forquantitative determination of arsenic in NEST oyster tissue.Table 3.6 Arsenic Concentration Found in NEST oyster tissueAnalyzed by ETAAS Certified Value[As], j.tg.g 14.5, 14.8, 13.9 14.0 ± 1.276Because a number of arsenic species, most of which exhibit different physical andchemical properties, have been reported to exist in the environment and in digests or extracts of environmental samples, the ETAAS conditions must be set so that the sensitivitiesfor all these compounds are equal. In order to evaluate whether the established optimumconditions resulted in equal sensitivities for different arsenicals, standard solutions ofarsenobetaine (AsB), arsenocholine (AsC) and the tetramethylarsonium ion (TMAsj, wereanalyzed. These compounds were selected because of their presence in marine organisms.Their determination using ETAAS was investigated by using the optimized experimentalconditions previously established. Inductively coupled plasma-mass spectrometric (ICPMS) analysis was also performed on these solutions for comparison purposes (Table 3.7).Data from the table indicate excellent arsenic recovery for the three arsenicals analyzed.Table 3.7 Arsenic concentration of solutions containing organoarsenicals[As}, [tg.mLCompound ETAAS ICP-MSArsenobetaine 0.065 ± 0.003 0.069 ± 0.003Arsenocholine 0.054 ± 0.002 0.058 ± 0.003TetramethylarsoniumIodide 0.050 ± 0.00 1 0.050 ± 0.003± Sample standard deviation, n=3.From this work, it becomes apparent that the application of an optimization proceduresuch as simplex (in this case the CMS method), in conjunction with a SRM, is of particularvalue when it is necessary to determine concentrations of a metal or a metalloid such asarsenic in environmental samples. Optimum conditions for various sample matrices can bequickly reached, thus eliminating matrix effects and also sensitivity variations resulting fromthe different arsenic species present in a particular sample.77This optimization procedure may also be used to improve the analysis of arsenic in thepresence of various reagents or buffers used in HPLC eluents, e.g. ion-pair reagents such asheptanesulfonic acid.Recently a number of reports have been published on the analysis of arsenic by usingETAAS in conjunction with mixed modifiers, e.g. palladium and magnesium.’74 Theprocedure used here would be ideal for optimizing the concentrations of these modifiers inconjunction with the appropriate furnace heating program.We believe that the optimization procedure used here can easily be applied to intelligent automated ETAAS optimization. Since most ETAAS spectrometers can beprogrammed, the addition of simplex optimization is feasible. This would then allow for theoptimal analysis of a great variety of environmental samples without any great knowledgeabout the sample matrix and its effects on arsenic absorption.3.3.2 Determination of Arsenic in Environmental Samples by using HGAASThe first hydride generation method to gain acceptance, was that introduced byHolak.’75 This method employed a metal (Zn) / acid (HCI and/or H2S04) reductionmixture to generate hydrides. A number of other metals, such as, aluminum powder,176 ormagnesium metal and titanium(III) chloride, have also been used to generate arsines inconjunction with an acid.57J48 This metallacid reduction technique has a number ofdisadvantages, which have limited its current use. These include: slow reaction times, up to20 mm,57 and difficulty in automating the system because of the metal addition. As aconsequence only As, Sb, and Se hydrides have been examined by using this method.HGAAS methods based on the production of hydrides by using sodium borohydride /acid reduction mixtures have been used with great success for the analysis of arsenic andother hydride forming elements since 1972.177 This reduction procedure has allowed forthe automation of HGAA systems. In the present work a continuous flow system was used78(Figure 3.2). This system has been previously optimized for the analysis of arsenic by Cullenand Dodd. 178The arsine generation reaction (Eq. 3.1) and the decomposition of the excess sodiumborohydride (Eq. 3.2) occur simultaneously.As(OH)3 + BH4 + H > AsH + H3B0 + H2 (Eq. 3.1)BH4 + 3H20 + H > HBO + 4H2 (Eq. 3.2)Although many arsenic compounds can be reduced to their corresponding arsines,only arsenite, arsenate, methylarsonate, dimethylarsinate, and trimethylarsine oxide arereadily converted to volatile hydrides upon reduction with sodium borohydride under acidicconditions. In order to use HGA.A to determine the total arsenic concentration in sampleswhich contain arsenic compounds other than thOse mentioned above, a digestion methodmust first be used that is capable of converting all arsenic compounds into hydride formingarsenicals. In this work we have evaluated a number of digestion methods that are used todigest environmental samples prior to their being subjected to arsenic determination byusing HGAAS.3.3.2.1 Wet Digestion HGAA Determination of ArsenicFour mussels (Mytilus cahfornianus) were blended, homogenised and subsequentlyfreeze dried. Four sub-samples of the freeze dried homogenate were wet digested andanalyzed for arsenic by using HGAA. The result obtained was 12.9 ± 0.6 ppm arsenic. Fourmore sub-samples of the mussel homogenate mentioned above were also wet digested andsubsequently treated with 1% KI 15 mm prior to the HGAA analysis. The concentration ofarsenic present was found to be 12.8 ± 0.5 ppm. The iodide in this case was added in orderto prereduce As(V) to As(III), prior to the NaBH4 reduction step. Several reports have79shown that As(V) usually exhibits lower sensitivity in the HG process than doesAs(III), 179-181 probably because of their different rates of reduction. These differences areespecially pronounced when peak heights are compared. Peak area does not, seem to beaffected by the oxidation state of arsenic in the solution. Our results indicate that theaddition of KI is not required for the complete recovery of arsenic in mussel samples. Thisis most likely a consequence of the measurement mode. Hydrides were continuouslygenerated and swept into the optical path of the spectrometer, and measurements weretaken only after the absorbance signal had stabilized.These mussel samples were also analyzed by using neutron activation analysis. Thismethod gave an average result of 11.4 ± 0.4 ppm arsenic.Standard reference materials were also analyzed. After wet digestion, DOLT-i wasfound to contain 9.4 ± 0.5 ppm arsenic, the certified value for this material is 10.1 ± 1.4.DORM-i was also analyzed and was found to contain 15 ± 1 ppm arsenic, the certifiedvalue of which is 17.7 ± 2.1 ppm arsenic.Wet digestion HGAA was employed for the analysis of byssal threads fromCalifornian mussels. The byssal threads were found to contain 3.2 ppm arsenic.From the results reported above it can be concluded that the wet digestion HGAAmethod used for the analysis for arsenic provides excellent recoveries of arsenic frombiological samples of marine origin.3.3.2.2 Microwave Digestion HGAA Determination of ArsenicSRMs (0.2-0.4 g sample size) were microwave digested and then analyzed for theirarsenic content by using HGAA. A variety of reagent mixtures were used to convert theorganoarsenicals present in SRMs into forms which could be readily reduced to hydridesand subsequently detected by using HGAA. Table 3.8 lists a number of different reagentcombinations tested in this study, along with the arsenic recoveries obtained.80Table 3.8 Recovery of arsenic from DOLT-i and DORM-iVolume (mL) of reagent used________Duration ofHNO H9SOd H,O, radiation (sec)As can be seen from these data, this procedure generally resulted in very low arsenicrecoveries from the SRMs. The addition of H2S04 increased the recoveries, perhapsindicating that larger amounts of the sulfuric acid are needed for the completedecomposition of any organoarsenicals. We did not pursue these conditions further becauseof the possibility of pressure build-up in the bomb which could have hazardous effects: therewas also a possibility of melting the Teflon® digestion vessel. The highest arsenic recoveryobtained for DOLT-i was 52.5%, while for DORM-i it was 36.2%. Compared with thewet digestion HGAAS and microwave digestion ETAAS procedures, this method results invery low arsenic recoveries. Apparently the experimental conditions used for the microwavedigestion were not rigorous enough to decompose the organoarsenicals. This indicates thata substantial amount of arsenic present in the dogfish flesh is in a form which is not easilyreducible, which is in agreement with other studies that have reported 89% of the arsenic inDORM-i to be present as arsenobetaine,48’82a compound that has been found to be verydifficult to decompose. Raptis et al.’82 experienced similar low recoveries of arsenic whenattempting to decompose fish tissue extracts with nitric acid. Studies on Mytilus edulis(Blue mussel) have also reported low arsenic recoveries obtained by using this method: 1.5Arsenic recovery (%)DOLT-i DORM-i6 120 20.3 10.11 1 60 20.1 14.42 2 120 23.9 15.42 2 i20+120 24.2 15.61.5 0.5 90 27.3 17.11.5 1.0 90 46.8 28.61.5 1.5 90 52.5 36.21.0 1.0 i.0 90 47.1 30.781ppm arsenic (wet weight) when analyzed by using microwave digestion-HGAAS and 2.8ppm arsenic analyzed by wet digestion-HGAASJ83The excellent arsenic recoveries obtained after analyzing the microwave digestate byusing ETAAS reinforce the idea that incomplete decomposition is the reason for lowrecoveries when using HGAA.3.3.3 Comparison of Methods used for the Determination of Arsenic inEnvironmental SamplesA typical calibration plot for arsenic, obtained by using ETAAS is shown in Figure3.5. Linearity is typically observed in the range of 0.2 to 3.0 ng arsenic. The absolute limitof detection (LOD), defmed as the amount of analyte giving a signal equal to the blank plusthree times the standard deviation of the blank, was determined to be 0.1 ng arsenic. Therelative standard deviation (RSD) of twenty injections of 0.4 ng arsenic was calculated to be5.6%. In most cases 20 iL sample solutions were injected into the graphite furnace.0.320.28,-... 0.240.20‘ 0.160.120.080.04_________0.000.00 0.01 0.02 0.03 0.04 0.05Arsenic Conc. (p.p.m.)Figure 3.5 Typical calibration plot obtained by ETAAS. Dotted line indicates 95%confidence limits; r: correlation coefficient; b: slope; a: intercept; 5: standard deviationr=0.999b=8.2a=0.015S =0.006y/xSb = 0.3S =0.006&820.120.10‘ 0.080.060.040.020.000.00 0.01 0.02 0.03 0.04 0.05Arsenic Conc. (p.p.m.)Figure 3.6 Typical calibration graph obtained by HGAA. Dotted line indicates 95%confidence limits; r: correlation coefficient; b: slope; a: intercept; S: standard deviationIn HGAA, approximately 5 mL of sample solution is required for each determination.This method has a LOD of 0.5 ng.m11 for arsenic, or an absolute LOD of approximately2.5 ng arsenic. A typical calibration plot for this method of arsenic analysis is presented inFigure 3.6. These plots are typically linear up to 100 ng.mL arsenic. The RSD of twentydeterminations of a 20 ng.mL1 arsenic solution was 3.1%.In comparing the two methods that are used for the analysis of arsenic inenvironmental samples, their advantages and disadvantages are apparent. ETAAS exhibits amuch better absolute LOD (0.2 ng) and thus is best suited for the analysis of samples forwhich only small volumes of solution are available. HGAA has a much better LOD in termsof arsenic concentrations (0.5 ng.mLl), and thus is better suited for samples of largervolumes (>15 mL). One of the main advantages of HGAA is its inherent ability to eliminatemost interferences, since the arsine products are separated from the sample matrix.83Interferences are a major problem for ETAAS, thus the experimental conditions used forthe analysis must be carefully optimized in order to remove such problems.HGAAS can easily be coupled on-line to HPLC184 or to a digestion method e.g.microwave digestion.64 This is a very difficult procedure to apply to ETAAS and has notbeen used to any great extent. There are major limitations on the chromatographicconditions that can be used, because of the requirement for very low flow rates.73The main disadvantage of using HGAAS to determine total arsenic in marine samplesis that all organoarsenicals must be decomposed to arsenic compounds which are readilyreduced to volatile arsines.185 As has been demonstrated in this work, this decompositionis not always easily accomplished, and vigorous digestion methods are necessary. ETAAS isnot as dependent on the digestion procedure used, because the ashing stage of the heatingprocedure acts as a very efficient digestion step.Several other analytical techniques have also been used for the analysis of arsenic.Inductively Coupled Plasma-Mass Spectrometry (ICP-MS)48J86 is now a widely usedtechnique and is similar to ETAAS in that it only requires sample dissolution prior toanalysis. The two techniques differ in relative ease and cost of operation, and the types ofinterferences encountered. 1 72,173 Other less sensitive analytical techniques such asNeutron Activation Analysis, and ICP-atomic emission spectroscopy, have also beenemployed to determine total arsenic.1353.3.4. Arsenic Concentrations in Californian Mussels Collected from the B.C. CoastBivalve mussels have been used in many marine environmental programs, as in situbioindicators of trace metal exposure. However, it is very difficult to assess the origin ofarsenic, as being natural or anthropogenic, by merely determining the total arsenic contentof the animal.84The main purpose in analyzing marine animals for their arsenic content is forregulatory purposes, because these animals constitute a major human food source. Suchdeterminations enable estimations to be made of arsenic uptake by humans. For example,the daily intake of arsenic in Japan is estimated to be 70-170 jig.day-1,87,188 while it is 7-60 j.tg.day in countries such as Canada, UK, USA, and France.189The arsenic concentrations in the Californian mussel collected from a variety oflocations are listed in Table 3.9. The results were obtained by using the two digestion anddetection methods discussed in detail in this Chapter. All concentrations are quoted on a dryweight basis. Three replicate analyses were carried out for each sample.Table 3.9 Concentration of arsenic in Californian musselsLocation on British No. of mussels As (jtg.g1)Columbia Coast pooled Wet Dig. - Microwave Dig. -HGAA ETAASQuatsino Sound 5 14.1± 0.5 14.5± 0.5(Koprino Bay)Quatsino Sound 5 12.9± 0.6 13.1± 0.5(Varney Bay)Anthony Island 6 9.6± 0.6 9.2± 0.4Langara Island 6 10.1 ± 0.3 10.9 ± 0.5Tofino 7 15.2 ± 0.6 15.6 ± 0.6China Beach 4 11.4 ± 0.5 12.2 ± 0.6To the best of our knowledge these are the first reports of arsenic concentrations inCalifornian mussels, thus the levels can only be compared with those of other marineanimals. In general the values in Table 3.9 are close to the average values reported for othermarine species. Blue mussels (Mytilus edulis) for example, are found to contain 1.8 ± 0.4.tg.g1 arsenic, on a wet weight basis.’78 This is equivalent to approximately 14 -16 j.tg.g’arsenic, on a dry weight basis, It should be noted that the highest arsenic values came from85mussels originating from Quatsino Sound close to a copper mine, and from Tofino, an areawhere anthropogenic arsenic input is considered very low.Arsenic distributions in the various tissue parts of the Californian mussel were alsoinvestigated in order to determine whether any particular mussel organ concentrates arsenicpreferentially. For this analysis, five mussels were dissected and their tissue parts werebulked together. Table 3.10 shows the data obtained for such determinations.Table 3.10 Arsenic distribution in Californian mussel tissue partsTissue Part As (ig.g4)Muscle 7.0±0.9Visceral mass 12.5 ± 05Gill 10.4±0.6The highest arsenic concentration was found in the visceral mass of the mussel.Studies on Mercenaria mercenaria (clam) and Mytilus coruscum have shown similararsenic distributions.50”9 In contrast Shiomi et al.,28 showed that the gill of the clamMeretrix lusoria contains higher arsenic concentrations (21.5 ig arsenic g fresh tissue)than other tissue parts (>5.4 jig arsenic g1 fresh tissue). Similar trends were reported byShibata and Morita5°for the same clam species.863.4 SUMMARYSimplex optimization was used to efficiently delineate the optimum experimentalconditions to be used for the ETAAS analysis of arsenic in a SRM of marine origin, whichhad previously been microwave digested. Four experimental variables, were considered:ashing temperature, atomization temperature, modifier concentration, and atomizationramping time. This combination of methods and materials provides a powerful means ofrapidly improving the experimental conditions used for the analysis of arsenic in a widevariety of samples of environmental origin. Excellent recoveries of arsenic were obtainedwhen using the optimum electrothermal atomic absorption spectrometry conditions toanalyze standard solutions of arsenobetaine, arsenocholine and tetramethylarsonium iodide.Other methods used to determine arsenic in environmental samples of marine originwere evaluated. HGAAS showed very good arsenic recoveries when used to analyze SRMswhich had been previously wet digested. Microwave digestions (acidIH2O)followed byHGAA analysis did not offer acceptable arsenic recoveries.Finally Californian mussels collected from the B.C. coast were found to contain arsenicin the range 9 to 16 .tg.g4 dry weight basis.87CHAPTER 4DEVELOPMENT OF HPLC AND MS METHODS FOR THESEPARATION AND DETERMINATION OF ARSENIC ANIMALFEED ADDITIVES AND THEIR METABOLITES4.1 INTRODUCTION4.1.1 Scope and Rationale of WorkThe work reported in this Chapter is primarily concerned with the development ofanalytical methods capable of separating and determining organoarsenicals used in animalfeeds, as well as identifying their metabolites. Currently four arsenicals are approved as feedadditives for domestic animal production: 3-nitro-4-hydroxyphenylarsonic acid (3-NHPAA), p-arsanilic acid (p-ASA), 4-nitrophenylarsonic acid (4-NPAA), and pureidophenylarsonic acid (p-UPAA).191193 The development of analytical methodology isnecessary in order to provide information about these compounds that will enable us tounderstand their interactions and fate in the environment. In addition these methods wouldallow us to identify and quantify arsenic feed additives and their metabolites. This is ofparticular interest since there is very little information in the scientific literature, regardingthe arsenic compound interactions and/or their metabolic fates in the environment. Inaddition analytical procedures developed here could be utilized for pharmacologicalinvestigations.So far only a number of methods have been developed for the analysis of this class ofcompounds. Most of these, however, are for target analysis of a few specific compounds88and are not suitable for the detection of possible metabolites. For example gaschromatography (GC) with flame ionization detection (FID), which has been used for thedetermination of p-UPAA and p-ASA, is not suitable for the determination of 3-N}{PAAand is also interference prone.194 A spectrophotometric method for the determination of pUPAA in Carbarsone (proprietary formulation containing 33.6% w/v p-UPAA) has alsobeen reported.195 This method involves a coupling reaction with N-1-naphthylethyle-nediarnine. The coloured product that forms is extracted with butanol and subsequentlymeasured photometrically; again interferences can cause difficulties in this determination.Thin layer chromatography (TLC) has been used for the separation and identification of 3-N}IPAA, p-ASA, 4-NPAA, and p-UPAA, using colouring reagents for the visualization.’96HPLC has also been used to separate some arylarsenicals, but not specifically those used asanimal feed additives.178,197,1983-NHPA4Figure 4.1 Structures of arsenic compounds used as animal feed additives.The four compounds used commercially as feed additives are very polar. Thus, in thiswork NPLC was selected for study as being probably the most efficient method forachieving their separation, while GC was not considered because of the low volatility of thecompounds. Suitable derivatization could improve their volatility but may not allow for theHO-p-ASA 4-NPAA p-UPAA89detection of possible unknown metabolites which may occur. Micro-HPLC separation wasalso evaluated and compared to other conventional and microbore-HPLC systems. Themicro-LC columns used in this work were constructed and packed in our laboratory as partof continuing studies on the applications of these techniques, which appear to have manyadvantages.199 Ultraviolet (UV), ETAAS, Thermospray MS, Liquid secondary ion massspectrometry (LSIMS) and dynamic or continuous flow (CF) LSIMS were used asdetectors in conjunction with HPLC both in on-line and off-line modes.4.1.2 Arsenicals used as Animal Feed AdditivesThe use of arsenic in modem human medicine started in 1907 when Dr. P. Erlichdiscovered Salvarsan.20°This arsenic compound was used as an anti-syphilis drug andrelated arsenicals were developed for other medicinal applications. In 1930 Morehouse etal.201 were able to identifj copper arsenite as the active ingredient present in poultrydrinking water medication used for the control of coccidiosis. This marked the beginning ofstudies of inorganic arsenic compounds with regard to their ability to control coccidiosis;however these compounds proved to be too toxic for any practical application. Theseresults directed researchers back to the organoarsenicals that had previously been used inhuman medicine. In 1945 Morehouse showed that 3-NHPAA was capable of controllingcecal coccidiosis in poultry.202 While investigating the therapeutic performance of thiscompound it was also discovered that it acted as a growth promoter, providing growthstimulation, improved feed conversion, better feathering, increased egg production andpigmentation. In the following years a number of other compounds were shown to havesimilar properties, p-ASA, 4-NPAA, p-UPA.A and benzenearsonic acid. With the exceptionof the last one, all of these compounds are still in use today. Variation of the substituents onthe aromatic ring results in differences in the growth-promoting and disease-controllingeffects of the compounds. Thus, 3-NHPAA and p-ASA are approved as animal feed90additives for both poultry and swine, whereas 4-NPAA and p-UPAA are approved only forcontrolling blackhead disease in turkey.19’-193Recent studies however, have disputed the beneficial effects of these compounds asgrowth promoters. p-Arsanilic acid was found to be ineffective in improving the biologicalor economic performance of broiler chicks when added to their diet at the maximumpermissible level in Canada.12°This was also the case when 3-NHPAA was supplementedin the diet of growing-finishing pigs.203A number of reports have pointed out the absence of microscopic changes in tissuesfrom animals whose diets had been supplemented with these compounds, within theallowable levels. 120,203If these feed additives are used it has been well documented that, shortly after thearsenicals are removed from the animal diet, they are rapidly metabolized and depleted fromthe animal and within five days the levels of arsenic in the various animal tissues return tobackground levels. Thus the use of the arsenical containing feed must be discontinued fivedays prior to the slaughtering of the animal.A number of reports have pointed out that these additives can cause toxic effectswhen used at levels higher then the recommended levels. The maximum limit for 3-NHPAAis 50 mg per kg of feed, while the recommended level is 37.5 mg per kg. Reports show that400 mg of 3-N}IPAA per kg of feed when given to weanly pigs for 4 weeks resulted in 65%of the animals showing neurological signs:204 200 mg of 3-NRPAA per kg of pig feed didresult in weight gain; however, muscle tremors were also observed.205 Even 105 mg of 3-NHPAA per kg of pig feed resulted in toxic effects.206In order to evaluate the physiological role of these compounds as well as to assesstheir impact on the environment a number of areas need to be monitored for arsenic contentand arsenic species. Of main concern are: the producer who is exposed to the additives inthe feed mill, animal excreta used as a fertilizer and as a nutrient source in animal feeds, and91finally the effect on the human population both from a meat consumer point of view as wellas from considerations involving sources of drinking water (aquifer).4.1.3 Micro - HPLC used for the Separation of Arsenical Animal Feed AdditivesA great deal of research effort has gone into the development of microscale HPLC.This began in 1967, when Horwáth and co-workers used a 1 mm ID and 2 m length columnto separate nucleotides.207 Further development came in 1973, when Ishii and co-workerssuccessfully separated polynuclear aromatic hydrocarbons on a 0.5 mm ID x 150 mm lengthcolumn.208 There are a number of advantages associated with this particularchromatographic technique and these are the main reasons for continuing research in HPLCcolumn miniaturization. Some of the advantages are the following:First of all, the low consumption of both stationary and mobile phases, which is adirect consequence of the decrease in column dimensions, allows the use of expensivepacking materials, and exotic mobile phases, such as deuterated solvents. The techniqueallows for a dramatic reduction in solvent waste, which is a serious concern since today itcosts more to dispose of some solvents than to purchase them.Another advantage of the method is the increase in mass sensitivity when using aconcentration sensitive detector. This is a consequence of the reduced dilution of the sampleinjected onto the LC column, compared with that in conventional HPLC.Finally, the one feature that made microscale ITPLC especially appealing for thepresent application, is the ease with which it is coupled to a mass spectrometer. The latter isa direct consequence of the extremely low solvent flow rates employed in micro-HPLC,typically in the range of 5 - 20 i.tL.min for columns, that can be adequately handled bymost MS systems.Microscale HPLC columns can be classified into three categories depending on thephysical type of stationary phases and the packing states of the columns. Table 4.1 lists the92types of columns together with their specific characteristics. As can be seen the maindifference between densely packed and loosely packed columns is their column-particleratio (d/d). Open-tubular columns are unique in that their stationary phase is a coated orchemically bonded thin film on the inner surface of the capillary.Densely packed columns can be further classified according to their column innerdiameters into three categories: conventional HPLC with 4-6 mm ID columns, semi-micro(or microbore) HPLC with 1-2 mm ID columns, and micro-I{PLC with 0.2-0.5 mm ID.This classification is employed in the remainder of this Chapter. Most of the work presentedhere has been conducted on micro-HPLC columns (0.32 mm ID), densely packed with 3 or5 p.m diameter particles.Table 4.1 Classification of Microscale HPLC19920921°Common names of Column LD. Particle Differences indifferent types of (jim) diameter (jim) d I d packing state ofmicroscale HPLC kId Id1 columnsDensely packedcolumns250-1000 3-30 50-200Loosely packedcolumns50-200 10-100 2-8Open-tubular(capillary) columns10-60 Coated film934.1.4 Interfacing Liquid Chromatography to Mass SpectrometryAs mentioned in detail in Chapter 2, MS is one of the most widely used analyticaltechniques used for the detection and structural elucidation of organic, inorganic, andorganometallic compounds. Thus it is highly desirable to couple this extremely powerfuldetector to a separation technique as efficient as HPLC. For over twenty years combinedgas chromatography - MS has been applied with great success to the analysis of volatilecompounds. Consequently much effort has been devoted to applying combined LC-MS tothe analysis of polar and non-volatile compounds.The primary difficulty encountered in the early development of LC - MS interfaceswas the major difference in the operating environments of HPLC (liquid under pressure)and MS (sample and eluent vapour under high vacuum). As an example, consider anaqueous reversed-phase LC eluent flow at 1 mL.min’ which generates 1-4 L. In order toovercome such experimental difficulties many different types of LC - MS interfaces havebeen developed, some of which have become commercially available.The first report of a commercial LC-MS interface was a moving wire interface, inwhich the LC effluent was deposited on a heated wire. After the removal of the solvent thesample residue was transported into the ion source of the mass spectrometer.2A similardevice, called the moving belt interface (MBI), was more successful and is stillcommercially available.212 Eluent is sprayed onto a moving belt, via a heated nebulizer.The belt then passes under infrared heaters and subsequently through a series of vacuumlocks, removing all the solvent. Once the sample reaches the ion source it is flashevaporated off the belt and into the ionization chamber. This technique is compatible withEl, CI, and FAB ionization modes, and can handle non-volatile buffers. Its maindisadvantages are sample carryover (memory effects) and mechanical complexity.Another very interesting approach to solving the LC-MS interface problem is theDirect Liquid Introduction (DLI) method, pioneered by Tal’ Rose et a!.213 and Baldwin94and McLafferty.86 Typically, a solvent jet is formed by passing the liquid at a flow rate of5-20 i.tL.min1 through a 2-5 gim diameter hole in a replaceable diaphragm located at theprobe tip placed in the ion source. The ionization involves chemical ionization from themobile phase vapour. This method is restricted to low liquid flow rates, and can onlyemploy volatile buffers. These low flow rates are of course directly compatible with microHPLC methods suggesting a coupling of micro-HPLC and DLI. This is the approach wehave pursued in order to interface our micro-HPLC columns to a mass spectrometer.In contrast to the DLI interface, the thermospray (TSP) interface2l4,14vaporizesthe LC effluent just prior to its introduction into the ion source. This, in addition to auxiliarypumping, makes sampling possible at high solvent flow rates, up to 2 mL.min1. Aresistively heated stainless steel metal capillary is used to produce a jet flow, which consistsof fine mist droplets of column eluent. This jet is carried at a high speed into the heated ionsource, where it continuous to vaporize. The mobile phase contains volatile buffers(NET4OAc, TFA). The buffer ions form charged droplets which undergo evaporation andlead to production of sample ions in the gas phase. Sample and buffer ions are directed intothe mass analyzer, while the excess vapour (both solvent and sample) is pumped away by anauxiliary vacuum pump. The development of this method has reached a plateau and is nowin the hands of users throughout the world, as demonstrated by the significant increase inthe number of application reports in recent years.74’215Dynamic or CF FABJLSIMS, developed in the mid 1980s, has shown great promisefor LC-MS interfacing.2’6,217 The technique is well suited for the analysis of large, polar,and/or thermally labile molecules. Flow rates of approximately 5 iL.min are used with 1-10% matrix (glycerol, nitrobenzylalcohol, etc.) incorporated into the LC eluent. A flit isnormally placed at the end of the LC transfer line situated inside the ion source. This allowsfor sample dispersion and also provides for an impact area upon which the FAB/LSTMSbeam can be focused.95Electrospray (ES) I Ionspray MS is the most recent technique which functions as bothan LC-MS interface and ionization source.218’9ES operates in the continuous flow (CF)mode, as does CF-FAB, thus the sample is introduced and ionized in the liquid phase. Thismethod has undergone tremendous growth in recent years, and is evolving into thedominant LC-MS interface for a variety of applications. The basic principle of operation ofthis method is the formation of a fme spray of charged droplets in a strong electrostaticfield at the exit of a narrow capillary through which the solution of the analyte passes.Subsequent evaporation of the solvent allows for the formation of single and multiplycharged analyte ions. The exact mechanism of ion formation by ES is still under intensediscussion and investigation.220-24.2 EXPERIMENTAL4.2.1 Instrumentation4.2.1.1 HPLC pumpsTwo different types of pumps were used in these experiments.A Waters 510 reciprocating pump was used for the conventional HPLC columns. Adual syringe pump (Applied Biosystems, Model 140B) was used to provide the appropriatemobile phase gradient and flow rate for the microbore and micro columns. This systemconsists of two independently-driven syringe pumps with a volume of 10 mL each. A 2001iL mixer, used to form gradients, is also part of the system. This pump is capable ofdelivering pulse-free solvent flows at rates as low as I iiL.min4.Pulse-free mobile phaseflows are extremely advantageous for obtaining stable ion beams for the CF-FABILSIMSand ES methods.964.2.1.2 InjectorsSamples were introduced onto the conventional column via a 20 tL stainless steelloop injector (Rheodyne 7125). This dual-mode injector was operated in the complete-filling mode, in order to improve accuracy and precision of the sample volume loaded ontothe column. A 0.5 jiL internal-chamber micro injector (Rheodyne 7520) was used forloading samples onto microbore and micro columns.4.2.1.3 HPLC ColumnsThe conventional HPLC column used was a C18 Spheri-5 RP-18 (Brownlee Labs)with the following dimensions: 100 mm length x 4.6 mm ID, containing 5im diameterpacking material.Microbore separations were achieved on a C18 Spheri-5 RP-18 (BrownLee Labs),250 nmi length x 1 mm ID, containing 5 I.m diameter packing material.The micro columns in a variety of lengths (8, 14.5, 20 cm length) and 0.32 mm IDwere fabricated in-house and packed with C18 packing material (Spherisorb ODS2, 3 and 5.tm diameter).4.2.1.4 HPLC DetectorsFor the initial evaluation of the LC columns and mobile phases a UV detector(Waters, Lambda-Max Model 481) equipped with a 1 .tL flow cell was used to monitor theseparations, and allow for optimization of their conditions.A Kratos Concept II H mass spectrometer was used for the LSIMS and CF-LSIMSstudies. For the DLI-MS experiments a Nermag RiO-lOB quadrupole mass analyzer,equipped with a desolvation chamber and a cryopump, was used. A Kratos MS 80 massspectrometer was used for the TSP-MS.97The ETAAS system, used for the analysis of the LC fractions for arsenic, is describedin detail in Chapter 3 of this Thesis.4.2.1.5 LC-MS InterfacesIn order to perform micro-LC-MS experiments three interfaces were evaluated, a CFLSIMS probe and two different DLI couplings.The CF-LSIMS probe (Figure 4.2), located inside the high vacuum ion sourcechamber, delivers the mobile phase via a quartz capillary tube to a mesh screen at the tip ofthe probe. The quartz capillary is in firm contact with the mesh, thus allowing the liquid tospread over the mesh by vacuum and capillary actions. A porous steel body in contact withthe mesh acts as an absorbent for the excess solvent, and thus prevents potential peakbroadening caused by the probe tip. The latter is in contact with the heated ion source blockallowing for efficient heat transfer to the mobile phase, thus leading to its stable rate ofevaporation. This feature is critical for maintaining a stable ion source pressure and ioncurrents. +Cs beamFine wire meshPorous metal bodyLiquid delivery lineI__High voltage SS probe shaftinsulatorFigure 4.2 Kratos continuous flow LSIMS probe and Cs ion beam.Two DLI interfaces were used in these experiments (Figure 4.3). The first DLI probe(Fig.4.3A) was equipped with a nickel diaphragm having a 2-5 .tm orifice. A liquid jet wasIon extractionto mass analyzer98formed by forcing the mobile phase to pass through this orifice. The probe was inserted intoa heated desolvation chamber attached to the ion source. This allowed for the subsequentdesolvation of the liquid jet droplets.The second DLI interface (Fig. 4.3B) was constructed in-house. This interface issimilar in principle to a thermospray nebulizer, the main difference being its microdimensions. A quartz capillary (10 urn ID) is heated, thus converting the liquid mobilephase into a vapour jet, which is introduced into the mass spectrometers ion source. Forboth DLI interfaces an electron beam was used to generate ions for subsequent massanalysis.4.2.2 ChemicalsStock solutions of 1000 ppm of each of the following organoarsenicals wereprepared: 3 -nitro-4-hydroxyphenylarsonic acid (ICN Biochemicals, Cleveland, OH 44128),p-arsanilic acid (EASTMAN Organic Chemicals, Rochester 3, NY), 4-nitrophenylarsonicacid (Aldrich Chemical Co., Milwaukee, Wisconsin), and 4-hydroxyphenylarsonic acid(EASTMAN Kodak Company, Rochester, NY).All solvents (HPLC grade) were filtered and degassed prior to their use as mobilephase by using a 35 mm all glass filter holder fitted with an HA 0.45 urn filter (Millipore)for the aqueous solvents and a FH 0.5 urn filter (Millipore) for the organic solvents.99A.Mobile phaseI/ProbeB.‘1DiaphragmHeated desolvation chamberSStubeacusffiea pillaiyFigure 4.3 DLI nebulizers; A. diaphragm nebulizer with desolvation chamber, and B.heated capillary nebulizer.sourceHeated Cu sleeve with thermocoupleMobile phase1004.2.3 Procedures4.2.3.1 Fabrication and Packing of Micro HPLC ColumnsThe fabrication procedure for the micro LC column is schematically outlined in Figure4.4. Fused silica columns [Al (lIP-ULTRA 2) with internal diameters of 0.32 mm were cutto various lengths. These columns were used as the main body of the HPLC column. A 1cm plug of silanized glass wool was inserted into the column, 1 cm from the end. This wasachieved by slipping a 2 cm piece of Teflon tubing over one end, and using a piece of quartztubing (0.290 mm O.D.) as a pushing rod to insert the glass wool. Fused silica tubing (B](0.050 mm I.D., 0.143 mm O.D., Polymicro Technologies) 50-60 cm long was used forcoupling the micro-HPLC column to the detector. In order to connect this line to the microHPLC column (A] a piece of fused silica tubing (C] (0.181 mm I.D., 0.290 mm outerdiameter, Polymicro Technologies) 2 cm in length was placed over [B], and cemented intoplace by using glue (Lapage #8). [B]+(C] were then inserted into (A] up to the glass woolplug. Glue was also used to keep these parts together. In addition a piece of glass tubing(D] 4 cm in length (Accupette pipets) was used to support this junction, by placing it overthe connections and gluing it into place. Prior to packing the constructed column wasallowed to dry overnight.In order to pack the micro-LC column a packing reservoir was needed, as shown inFigure 4.5. Silica based C18 packing material (Spherisorb 3 or 5 jim ODS2) was used toprepare a slurry by sonicating 3 0-40 mg of the material in 1 mL of carbon tetrachloride. Theslurry was then transferred into the packing reservoir by means of a dropping pipette. Themobile phase (acetonitrile / water 6:4) not mixable with the carbon tetrachioride was filteredand degassed prior to use. In order to achieve packing of the column, a pressure of about5000 psi was exerted by the pump, while delivering the mobile phase. The packing of thecolumn can be followed visually to completion.101Figure 4.4 Fabrication of the micro-HPLC column.[A]IEEE //: Silanized glass wool-//-1II[A+B+C]I I[B] [C]I___I: Glue (Lepage #8)[B+C][D][A]+ Glue102TubingPacking Reservoir-114’ SS Coupling40 mg of 3 pm C18 Spheiisorb00S2 Paddng Materiai hi 1.5 mLof Carbon tetracNoride.114’ SS Tubing(High Pressure)40% Graphatized Vespel® Ferrule1/16’ X 0.4 mm ID0.32 mm ID Capillaiy ColumnFigure 4.5 Schematic diagram of the apparatus used for packing micro-HPLC columns.4.3 RESULTS - DISCUSSIONA number of combinations HPLC techniques and detectors were evaluated in thiswork for use in the analysis of the animal feed arsenicals (Figure 4.6). Since we wereinterested in comparing the performance characteristics of various HPLC columns ofdifferent diameters and flow rates, it was necessary to evaluate the column and detector asone system, rather than each component separately. The main reason for doing this, is thata detector may influence the separation achieved on a particular type of column. Forexample, the separation efficiency of a micro column will be greatly degraded if it is1/16’ SS Coupling50 pm ID Quartz glass transfer line103connected to a UV detector equipped with a larger volume 14 .iL UV cell, rather than a 1tL UV cell, while the separation efficiency of a conventional column will not be affected ifconnected to a detector with a 14 j.iL cell. In general when coupling all components in amicro-LC system it is important to avoid dead volumes, stagnation volumes, etc.DLI-MSCF - LSIMSMicro Column C-18 :::::::: LSIMSSyringe PumpMicrobore Column c-i8 UVReciprocating—Conventional Column C-18ETAASPump Thermospray MSOff-line couplingOn-line couplingFigure 4.6 Methods used for the separation and identification of animal feed arsenicals.4.3.1 Separation and Determination of Arylarsenicals by using HPLC-ThermosprayMSThe separation of the compounds 3-NHPAA., p-ASA, and 4-NPAA on a conventionalC18 column (5 p.m particle size, 4.6 mm ID x 100 mm length) was monitored by using UVdetection. The optimization of this chromatographic separation was carried out by varyingthe methanol content of the mobile phase, and its flow rate. The resolution (R)[R= 12 — ti, t: retention time, Wb: base peak width], obtained from(Wb,1 +Wb,2)each experiment for 3-NHPAA and 4-NPAA, is presented in Figure 4.7 along with tworepresentative chromatograms.104Thermospray MS was also used for the detection of these compounds. The [M+H]+ion was the base peak in the mass spectra of all samples analyzed. Figure 4.8 shows the TSPmass spectrum of p-ASA, which is typical for all the arylarsenicals analyzed. The TSP massspectra of the other arylarsenicals are included in Appendix A. TSP-MS provided littleinformation concerning the analyte structure, but the molecular weight of the analyte caneasily be determined. However, structural information via fragmentation can be obtained byoperating the TSP ion source in the auxiliary filament ionization mode. In this mode theelectron beam, which is produced by the filament, is situated in the TSP jet and can give riseto analyte fragmentation. This mode of ionization is also of interest when high contents oforganic solvents are being used. In addition, a discharge can be generated in the source ofsome TSP-MS systems, leading to some fragmentation. Collisional-induced dissociation(CID) can also be used to generate characteristic fragment ions, if a MS-MS instrument isavailable. For this study only the TSP-MS was available.Operating temperatures are critical for optimum performance of TSP-MS. Bestsensitivity is obtained when sufficient heat is provided to completely vaporize the sample,without causing it to pyrolize or change structure. Other temperature settings such as thoseof the vaporizer tip (probe) as well as the ion source temperature also influence thesensitivity of the detector. This feature of TSP-MS somewhat limits the utility of themethod when it is used for the identification of unknown compounds, because its sensitivityis compound dependent and TSP ionization does not always take place. It has been ourexperience, and that of others,223 that TSP-MS is a very useful technique for targetanalyses, but not as valuable for the identification of unknown compounds. This is because aset of four temperatures, all of which greatly influence the efficiency of ion formation, mustbe optimized for each compound. In searching for unknown compounds it is almostimpossible to perform such an optimization.1050.0200.015 0.0160.012<00100.0080.005°L0.000__________________‘ 2 46802 46 0.000 II I I 0 2 4 6 8 10 12 14 16Tune (Tnft) TtAE (mm.)Figure 4.7 A: Three dimensional graph showing the variation of resolutiop between 3-NHPAA and 4-NPAA when the methanol content of the mobile phase and the flow rate arevaried. B: UV Q=254 nm)-chromatogram of arylarsenicals (22.5 ng As of eachcompound), 1 mL.min’ flow rate, 95% water (0.1% TFA) and 5% methanol. C: UVchromatogram (254 nm) of arylarsenicals, 1.2 mL.min1 flow rate, 75% water (0.1% TFA)and 25% methanol.1.60 A.Co 1.0)a,a:v.940.020p-ASA B. C.106The arsenicals studied here, both the animal feed additives and a possible metabolite(4-hydroxyphenylarsonic acid) of 3-NHPAA, give similar responses in terms of signal forthe same amount of compound, when using the same set of TSP temperatures. However,the two calibration curves shown in Figure 4.9 exhibit slightly different slopes, thus thesensitivities for the two compounds vary slightly. However, the variation is not sufficientlylarge to raise concerns about widely different response factors for structurally relatedarylarsenical feed additives under the LC-MS conditions used here.The linearity of the curves (Figure 4.9) supports the possibility of quantitation withina limited dynamic range. Clearly, the use of a suitable internal standard would greatlyimprove the precision of the method. The LOD in TSP-MS for these compounds wascalculated to be between 30 and 33 ng (1 .5-1.6 ppm) of arsenic, depending on thecompound analyzed.The results obtained in this work indicate that TSP-MS is a very attractive techniquefor the analysis of arsenical animal feed additives. The non-volatile nature of the compoundshas made their analysis quite difficult by using other conventional MS techniques, such asDCI (Figure 2.13) and El MS. This is due to the fact that the compounds pyrolyze in theion source prior to their desorption, and thus afford mass spectra with no molecular orquasi molecular ions.107I,.M/ZFigure 4.8 Thermospray mass spectrum of p-arsanilic acid. Temperature settings: Probe at129 °C, vaporizer at 185 °C, source block at 223 °C, and jet at 120 °C.1 .5e+ 007Figure 4.9 Calibration plot obtained by using Thennospray-MS for p-arsanilic acid and 4-nitrophenyl arsonic acid. The error bars represent the signal range obtained from threeinjections of the analyte.I,218[M+H]7.I.HO-As-OHc-..C,)Ca?Ca?-..a?4I21I.a,-x0a)01 .Oe+0075.Oe+0060.Oe+0000 5 10 15 20Amount of Arsenic (ng)251084.3.2 Separation of Arylarsenicals on a Microbore C18 HPLC ColumnThe first step towards the miniaturization of HPLC systems was the development ofmicrobore LC columns.207’8 We found that the arylarsenicals can be separated by usinga microbore column (Spheri-5 C18, 1 mm ID x 250mm length) at a flow rate of 80 .tL.min1 (Figure 4.10). The methanol content of the mobile phase has a pronounced effect on theresolution, as noted previously (Figure 4.7). Higher methanol percentage decreased theretention time of p-ASA and 4-NPAA, but also decreased the resolution. Good resolutionwas achieved by using 10-15% methanol. The separation efficiency of this column is quitesimilar to that obtained on the conventional HPLC column (Figures 4.10 and 4.7B).However, because of the reduced sample dilution on the microbore column, the LOD for pASA is 0.22 ng of arsenic, when using UV detection, compared to 1.9 ng of arsenic on theconventional column for the same detector. Another advantage we observed is aconsiderable reduction in solvent consumption, leading to economic and environmentalbenefits.4.3.3 Separation of Arylarsenicals on Micro C18 IIPLC ColumnsThe elution characteristics of the arylarsenicals on the micro-LC columns (Figure4.11) are similar to those observed on the conventional (Fig. 4.7) and microbore (Fig. 4.10)columns, both in terms of the overall time required for the separation and the efficiency ofseparation.As expected the LODs for UV detection were improved by one and two orders ofmagnitude respectively, over those obtained by using microbore and conventional HPLCcolumns, respectively. The LOD for p-ASA was calculated to be 0.02 ng of arsenic.Seven runs were carried out in order to evaluate the precision with which retentiontimes can be measured while using the micro-LC columns. The relative standard deviationof the retention times was found to be 0.9 % for p-ASA, 1.2 % for 4-HPAA, 1.6 % for 3-109NHPAA, and 1.4 % for 4-NPAA. These results indicate that arsenical identification can bebased on retention times if standards are available.In terms of solvent consumption, a typical arsenical separation requires 0.14 mL, 1.12mL, and 14 mL of solvent respectively, on micro, microbore, and conventional LC columns.This means that a 99 % reduction in the volume of solvent waste is achieved by switchingfrom conventional to micro-LC, and a 87.5 % reduction when switching from microbore tomicro-LC. These percentages can translate into extremely large savings, in terms of bothsolvent consumption, and solvent waste handling and disposal.The use of other types of chromatography for the separation of arylarsenicals has alsobeen reported. Maruo et at. 197 used a 0.5 mm ID column (ID between that of the microand microbore columns used in this study) packed with an anion exchange stationary phase,to separate three arsenicals, p-ASA and two other compounds that are structurally similarto the arsenic animal feed additives; o-arsanilic acid, and o-nitrophenylarsonic acid. Theresulting UV-chromatogram showed a separation that required over 40 mm and broadpeaks that had baseline widths between 5 and 10 mm. Others have attempted to separate thesame compounds on columns similar to those used by Maruo et at.,197 by using non-suppressed i6n chromatography, which resulted in co-elution of o-arsanilic acid and onitrophenylarsonic acid.198 Both these reports re-inforce the effectiveness of the LCapproach we have utilized in order to separate arylarsenicals, often referred to as ion-suppressed reverse phase LC.The micro-LC columns were also evaluated for their potential to separate anadditional arsenical, 4-hydroxyphenylarsonic acid (4-HPAA), which can be regarded as apossible metabolite of 3-NHPAA, on the basis of structural similarity. Figure 4.1 lB showsthat the compound is quite well resolved from its potential precursor.110o 2 4 6 8 10121418TtE (nIn3nm)-chromatogram of arylarsenicals (2.5 ng As of eachcolumn, 80 iL.min1 flow rate, 85% water (0.1% TFA) andFigure 4.11 UV (=254 nm)-chromatogram of arylarsenicals (0.8 ng As of eachcompound); micro-HPLC column, 10 iL.miir flow rate, 85% water (0.1% TFA) and 15%methanol. A: Compounds separated: p-ASA, 3-NHPAA, and 4-NPAA B: 4-hydroxyphenylarsonic acid added to the analyte mixture of A.p-ASA4-NPAA0Figure 4.10 UV (A=254compound); Microbore C1815% methanol.p-ASA B.A. 0.0200.0160.012e00080.0040.0000 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 18ThwlE () TRvE &flifl.)1114.3.3.1 Effects of Packing Procedure and Particle Size on the Quality of Micro-LCColumnsMicro-LC columns fabricated in-house were packed with a 3 and 5 J.tm diameterspherical porous silica based 18 stationary phase. No noticeable difference in resolutionwas observed for these two sizes of packings. Most HPLC applications use silica particleswith a 5 or 10 jim size diameter, while 3 im particles, even though commercially available,are only slowly being accepted by the chromatographic community. The main reason forthis skepticism is the requirement for spherical shaped particles which result in higherquality packed columns and which are difficult to prepare uniformly. The small diameterparticles, which are now more readily available, seem to be spherical.The slurry packing procedure we adopted was evaluated in both the downward andupward direction, because it is not yet established which approach gives the better results.In our hands the two procedures produced columns with similar performancecharacteristics.4.3.3.2 Effect of Column Length on the Elution Characteristics of the ArylarsenicalsColumn length has a profound effect on the separation efficiency of the micro-LCcolumn. Table 4.2 summarizes the resolution (R) obtained on columns of various lengthsfor two pairs of arsenicals. It is evident that the longest column (200 mm) provides the bestresolution, while the 145 mm long column also exhibits good resolution.Table 4.2 Summary of micro-Column length vs resolution (R) obtained for arsenicalsColumn Length (mm) R (p-ASA I 4-HPAA) R(3NI{PAA / 4-NPAA)80 unresolved 1.3145 1.4 2.2200 1.6 2.71124.3.3.3 Effect of Temperature on the Elution Characteristics of the ArylarsenicalsIt is quite easy to control the temperature of the micro columns. In our case, this wasaccomplished by immersing the column in a water bath; a thermocouple was used tomonitor the temperature. Separations were carried out over a range of temperatures, from18 0C to 57 °C and the effect of temperature is quite pronounced. Retention times aredramatically reduced (Figure 4.12), as are resolutions, when the column temperature isincreased. This we believe is a consequence of an increase in the interaction of the methanolin the mobile phase with the analytes. As mentioned earlier (Section 4.3.1), a similar type ofbehaviour is observed at constant ambient temperature, when the methanol content of themobile phase is increased.200 p-ASA• 4—HPAA5 15 V 3—NFIPAAv v4—NPAA10:70Column Temperature (°C)Figure 4.12 Effect of temperature on the arylarsenical retention times.4.3.4 Detectors used for Microscale HPLCBecause UV detectors provide limited structural information about compoundseluting from HPLC columns, it is necessary to develop other types of detectors with thisapplication in mind. In this work we have evaluated a number of detectors for their ability toprovide structural and/or elemental information concerning the arsenicals used as animal113feed additives. For example, MS techniques such as continuous flow FAB and DLI canprovide structural information, while element specific detectors such as ETAAS can be veryuseful for selectively detecting arsenic. All of these detectors have the potential to be usedeither off-line or on-line.4.3.4.1 ETAAS used as a Detector for Microscaie-RPLCETAAS was used in the off-line mode for the detection of arsenic compounds elutingfrom LC columns. To date there has not been a LC interface developed for ETAAS whichhas received general acceptance.224 This means that fractions of the LC eluent must becollected and subsequently analyzed. This method has been extensively used in conjunctionwith conventional HPLC columns,225 where the main disadvantage is the low sensitivity ofthe method due to dilution effects in the LC mobile phase. In addition chromatographicresolution is degraded. Both of these features make it very difficult to detect minor arseniccomponents in environmental samples. In this study we have compared the chromatogramsobtained by using conventional, microbore, and micro HPLC coupled off-line to an ETAAdetector.Fractions were collected every 30 sec from all three different types of column. Thisprocedure resulted in fraction volumes of 500 1.tL for conventional columns, 40 jiL formicrobore, and 5 1iL for micro columns. The resulting ETAAS-chromatograms are shownin Figure 4.13.The LOD for p-ASA on the three chromatographic systems was calculated to be 250ng of arsenic for the conventional column, 2.5 ng of arsenic for the microbore column and0.3 ng of arsenic for the micro column. Although the ETAAS detector is not concentrationsensitive an improvement in LOD for the micro column, was observed. This is expectedsince all of the LC eluent is injected into the graphite furnace while only 2 % and 37.5 % ofthe eluent from the conventional and the microbore column are injected, respectively.114Retentiono 2 4 6 8o 2 4 6 8RetentionTime (mm.)10 12 14 16 1814 16 18Figure 4.13 ETAAS-chromatograms of arsenicals, separated on a: A. conventional-LCcolumn, 500 j.tL fractions collected, B. microbore-LC column, 40 pL fractions collected, C.micro-LC column, 5 pL fractions collected.0.30.20.1oco0.150.100.05oco0.30.20.1Q)a.)0.010 12Time (mm)115The micro column has the disadvantage that the eluent volume allows only for a singleanalysis of each fraction, instead of two or three as is the case with the other two LCsystems. This translates into potential problems in the accuracy and precision of theanalysis. Of all three LC systems used, the microbore column seems to be the most suitedfor this application since its has a good LOD, and also allows for replicate determinations ofeach fraction collected.However, it should be noted that the LODs obtained in this study can be easilyimproved by performing multiple injections of each fraction into the AA furnace. Thedrawback of course would be a dramatic increase in the time required to analyze all thecollected fractions.4.3.4.2 Liquid Secondary Ion Mass SpectrometryFAB-MS has been recognized as a powerful tool in the mass spectral analysis ofpolar, thermally labile and/or involatile compounds. The operating principle of the methodhas been described in detail by Barber et a!. P226 In brief; the analyte is dissolved in aviscous matrix of low volatility such as glycerol, and subsequently introduced into the ionsource of the mass spectrometer, where it is bombarded by a beam of high energy (6-8 keV)neutral (> 80%) species (Ar, Xe). A similar technique termed liquid secondary ion massspectrometry (LSIMS) uses high energy Cs+ ions, instead of neutrals. The two methodsyield similar results, although some differences can result, since the primary ion beam isgenerally more focused compared to the neutral beam, is of higher energy, and usually has ahigher density of particles. Thus, in general LSIMS tends to be the more sensitive method.Spectra obtained in FAB and LSIMS are affected by the nature of the matrix227 and insome cases strong matrix-analyte interactions exist in the solution.228In this work we have utilized both LSIMS and continuous flow CF-LSIMS to analyzethe animal feed arsenicals. LSIMS has the potential to be used as an off-line LC detector,while CF-LSIMS can be used on-line.116In LSIMS a variety of different matrices namely, 3-nitrobenzylalcohol (NBA),glycerol, and thioglycerol, were investigated for their ability to promote ionization of thearsenicals. The mass spectra of 3-NHPAA, in both negative and positive ion detectionmodes, are presented in Figure 4.14: the matrix was NBA. The main feature of the positiveion spectrum is the formation of [M+H]+ ions and protonated reaction adducts of thematrix and the analyte.Negative ion mass spectra generally appear 1 Da below the molecular weight of thecompound being analyzed, because of the loss of a proton, which is required for negativeion formation, as opposed to a proton gain which results in positive ion formation.The triple ion pattern ([M+Hj+ ion, and protonated reaction adducts of the matrixand the analyte) observed in these spectra provides molecular weight informationconcerning the analytes. The general features of the mass spectra of all the arylarsenicals aresimilar to those observed for 3-N[TPAA in Figure 4.14. Appendix B contains the LSIMSmass spectra for these compounds.The use of CF-LSIMS to analyze the arsenicals is seen in the positive ion massspectrum obtained for p-ASA (Figure 4.15). In addition to the [M+H]+ ion and the matrixadducts, a number of characteristic fragment ions are also observed. The mobile phase, usedto deliver the analyte at a flow rate of 5 iiL.min’ to the ion source, consists of 89 % H20(0.1% TFA), 10 % CH3O , and 1% glycerol. These conditions were selected because theysatisfied both the requirements set forth by the micro-LC system used for the separation ofthe arsenicals and the LSIMS, which required the presence of a matrix-substance, such asglycerol, in order to perform the analysis. Micro-HPLC chromatography of the arsenicalsunder these conditions produced UV-chromatograms which showed few effects associatedwith the presence of 1% glycerol. However, some problems could be avoided byintroducing the matrix solution after the LC separation has taken place (post-column modeof introduction). 228a,b117•SIT(suo!XTJ1Wyg*)uOnpUOTAl1TSOJflpu‘UOnpUOTyydJ{s-jouiodsssuzswas’ivrvnth 1RelativeIntensity0-——*--:+:+:1-—--—I%1II’—+1’:+The concentration of glycerol matrix is much lower than that used in the conventionalstandard probe LSIMS conditions (described earlier in detail), and this considerably reducesthe signal from matrix ions (chemical noise). Another advantage associated with the CFmethod is that with time the resulting spectrum changes through the cycle, pure matrix tosample plus matrix to pure matrix, as the sample elutes. Again this permits backgroundsubtraction, and thus provision of clean mass spectra. The p-ASA spectrum shown in Fig.4.15 has been cleaned up in this way. The minimum detectable amount (M1)A) of p-ASAwas determined to be approximately 10 ng of arsenic, under the experimental conditionsused in this study.i:: [M+H1 p-ASA80706050•40 +[M+GIyc+H] [M.t-2G1yc+2H+MeOHJ20 .Hj4 MeOHL4 -H20 ao____10- 9’ [Cs] I 291 232 .2.. 417I 13 I I 254 292 i j.-f-4100 150 200 250 300 350 400 450 500Figure 4.15 Background subtracted CF-LSIMS mass spectrum of p-ASk mobile phase:89% water (0.1% TFA), 10% methanol, and 1% glycerol (Glyc); flow rate; 5 tL.min.1194.3.4.3 Direct Liquid Introduction- Mass SpectrometryDLI-MS is another mass spectrometric method with potential for coupling on-linewith micro-LC. Two different nebulizers were used in this study. A heated capillarynebulizer was built in house, while a diaphragm nebulizer with an associated desolvationchamber was obtained commercially.Use of the diaphragm nebulizer (Fig. 4.3A) enabled the production of mass spectra inthe negative ion detection mode. The main disadvantage of the method was its requirementfor relatively large amounts of analyte, compared to the other mass spectrometric methodsdiscussed in this chapter. Mass spectra were obtained with approximately 1000 ng ofarsenic, in the negative ion detection mode. As can be seen from the mass spectra of 3-NHPAA (Figure 4.16) and 4-NPAA (Figure 4.17), extensive fragmentation has occurredand the molecular ion is not the base peak as was the case with most of the other MSdiscussed methods before. This is in accordance with our previous observations that thesearylarsenicals are thermally labile and decompose/pyrolize upon extensive heating. Theheating in this case is provided by the desolvation chamber. To the best of our knowledge,this is the first reported use of this type of nebulizer for the analysis of organometalliccompounds.The heated capillary interface (Fig. 4.3B) was also tested. No meaningful spectra ofthe arsenicals were produced when using this nebulizer. Again, this can be explained interms of analyte decomposition, caused by the extensive heating which is required in orderto convert the CF-liquid into vapour. Organic test compounds, used for the initial set-up ofthis system, were analyzed successfiully. These included, caffeine, naphthalene, and 2,6-dimethyiphenol. The spectra of these compounds mainly consisted of [M+Hf ions (massspectra of these compounds are presented in Appendix C).120[M-0HJlOOx 246[Mi o_.263 II-..• HO-As-OH-..C1NO2C:1a?OHa?25>:213 233 It1111111111hi1H1L’ !IL.II.I IiiiI..Li,iJ tiII pi. .IlIiIll’208 221) 240 260 281) M/ZFigure 4.16 DLI-MS of 3-NHPAA, mobile phase consisted of 90% water (0.1 %TFA),and 10 % methanol, 2012L.min’ flow rate, negative ion detection.100>:- 213 [M-20HJ0. II75>: HO-As-OHa?C50>:• [M-O] NO2a? 231 [MJ247r ‘ .iuJk4, ji_fl flu, F-.150 170 190 218 231) 250 270 291) 37 M/ZFigure 4.17 DLI-MS of 4-NPAA, mobile phase consisted of 90% water (0.1 %TFA),and 10 % methanol, 20 L.min4 flow rate, negative ion detection.1214.4 SUMMARYThe work described in this chapter deals with the development of analyticaltechniques which can be used for the analysis of arsenic compounds added into animalfeeds. More specifically, chromatographic methods have been developed for the separationof these arsenicals. In particular micro-LC, which is a relatively new technique, shows anumber of advantages for these analyses, especially when interfaced to MS. Improved limitsof detection, low solvent consumption, lower use of stationary phase, as well as flow rateseasily accommodated by CF-LSIMS and DLI-MS, are some of the advantages we haveobserved when fabricating, and using these columns. Off-line coupling of micro-LC toETAAS improves the LOD for arsenic and opens the possibility of on-line coupling, an areawhich requires extensive research before it becomes reality.Additional advances could also be made in this area by coupling micro-columns toMS-MS systems or alternative detectors such as ICP-MS. These aspects of micro-LCremain to be evaluated for their effectiveness in analyzing not only arylarsenicals but alsoarsenic compounds encountered in the marine and terrestrial environment.Further use of the methods developed here will allow for a more detailedunderstanding of the fate and interactions of arylarsenicals in biological and environmentalsystems.122CHAPTER 5ARSENIC SPECIATION IN MYTILUS CALIFORNIANUS MUSSELS5.1 INTRODUCTIONAs mentioned in the General Introduction of this Thesis (Chapter 1), a number oforganoarsenicals have been reported to exist in marine organisms. Arsenosugars found inmarine algae, along with arsenobetaine (AsB) found in marine animals, are some of the mostsignificant arsenicals in this respect.113 Francesconi and Edmonds have proposed amechanism for the biosynthesis of the arsenosugars (Figure 5.1), and for the conversion ofarsenosugars into AsB (Figure 5.2).42,229It is believed that arsenate is incorporated by marine organisms, possibly because ofits structural similarity to phosphate,230 and subsequently methylated according toChallenger’s mechanism44(Figure 1.1), thus forming MAsA and DMAsA. According to theproposed mechanism (Figure 5.1) for the biosynthesis of arsenosugars, an arsenic-containing nucleoside (dimethylarsinyladenosine) is produced after S-adenosylmethionine(Figure 1.2) donates an adenosyl group to DMAsA. Dimethylarsinyladenosine has beenisolated from the kidney of the giant clam, Tridacna maxima, by Edmonds et a!. 30 Anumber of dimethylarsinyiribosides identified from algal sources may have resultedfollowing enzymatic hydrolysis of this compound. Although the order of alkylation in thebiosynthesis of dimethylarsinyiribosides shown here is the most likely, adenosylation mayprecede one or both, of the methylation steps.123Figure 5.1 Proposed mechanism for the biosynthesis of arsenosugars by marine algae.0OHReduction thenmethylation0IIMe—As—0OHMAsAReduction the,nmethylationDMAsA0 Reduction then 0II Methylation’9 IIMe2As—O Me3AsReduction thenadenosylation÷Me4AstReduction thenMethy1ation??Reduction thenAdenosylation2,c5MeSA_-\flHOOHGlycosidationMe3A ORReduction thenMethylation??I GlycosidationMe2L—\0,ORHOOHReduction thenMethylation??HO124It has also been proposed that trimethylarsonioribosides are formed following thismechanism, although again in this situation the stage at which the third methyl group istransferred to arsenic (in Figure 5.1 it is shown as the final stage) is still not clear. It ishowever, possible for the methylation of arsenic to proceed in algae without adenosylation,thus producing a tetramethylarsonium salt. This type of arsenical has not been identified inalgae, however it is a common constituent in bivalve mollusks. 1,28,231It has been suggested that AsB is a degradation product of the arsenosugars. Theproposed mechanism of conversion of dimethylarsinylribosides to AsB is outlined in Figure5.2A.13 This conversion requires the cleavage of the C3-C4 bond of the ribose ring,followed by oxidation at C4, reduction and further methylation. Limited experimentalevidence is available to support this proposed pathway.The identification of a trimethylarsonioriboside in a brown alga, Sargassumthunberg-ii, and in Tridacna maxima kidney, allows for the proposal of an alternativepathway for the formation of AsB, as shown in Figure 5.2B.13 The degradation of thetrimethylarsonioriboside to arsenocholine (AsC) under anaerobic conditions has been shownto occur with virtually quantitative yield.232 It has also been shown that AsC is convertedto AsB when fed to yelloweye mullet.2335.1.1 Scope and Rationale of WorkThe main purpose of work described in this chapter was to isolate and identifjarsenicals present in Californian mussels. A number of purification techniques were adoptedin order to carry out this task. Once these compounds were purified they were identified byusing mass spectrometric techniques, as well as by using high performance liquidchromatography (HPLC) coupled to atomic absorption spectrometry (AAS).125methylarsinylacetic acidA.ionAnaerobic 0DecompositionMe2As-CH—-CHO\sreduction then\,methy1ationDimethylarsinyi.ribosidesB.AnaerobicDecomposition Me3t—CH2-OH oXidation Me3A—CH2—-COOArsenocholine (AsB)Figure 5.2 Proposed mechanisms for the biosynthesis of arsenobetaine.5.2 EXPERIMENTAL5.2.1 InstrumentationA Varian Techtron Model AA 1275 Atomic Absorption Spectrometer equipped with aVarian GTA-95 accessory, was used to determine arsenic by using Electrothermal AtomicAbsorption Spectrometry (ETAAS). For these determinations a chemical modifier consistingof 500 ppm Pd in 2% citric acid was used. Chapter 3 of this Thesis provides details concerningthe method employed, as well as the conditions used for the ETAAS analysis of arsenic.HO0IIMe2As-CH--COOreduction then\iy1ationMe3AstCHTCOO(AsB),,,/ationMe3At—CH!—-CH2OArsenocholineOH126A quadrupole mass spectrometer (DelsifNermag RiO-lOB) incorporating a desorptionprobe, an electron beam ion source, a quadrupole mass filter, and a channeltron detector, wasused for the low resolution DCI analysis of the arsenic compounds. High resolution(R=10,000, 10% valley definition) DCI measurements were carried out using a doublefocusing mass spectrometer (Kratos Concept II H / Mach3 Data system). Details concerningthese two instruments as well as their operating conditions are given in Chapter 2 of thisThesis.The HPLC system used consisted of a Waters M45 pump, a Waters U6K injector, theappropriate column, and an automated fraction collector. Separations were achieved byusing two different columns, a Protein Pak DEAE 5PW column (7.5 mm I.D. x 75 mmlength, Waters), and a Bondclone 10 C18 column (3.9 mm ID. x 300 mm length,Phenomenex).The hydride generation gas chromatographic system used in this work has beendescribed in detail in Chapter 2 (Section 2.2.1.3, Figure 2.3). The only difference here isthat an AA spectrometer is used as the detector instead of a MS detector. The AAspectrometer used was an Instrumentation Laboratories 351 AAS. The atomization of thearsines took place inside a quartz cuvette, mounted in a hydrogen-air flame. The signal wasmonitored at 193.7 nm and processed by using a Hewlett-Packard 3390A integrator.5.2.2 ChemicalsThe origins of the synthetic arsenic compounds used for this investigation, have beenspecified in Chapters 2 and 3.5.2.3 Procedures5.2.3.1 Extraction of Organoarsenic Compounds from MusselsFrozen Californian mussels, collected from Vancouver Island (China Beach, Sooke),were thawed and their shells were removed. This procedure resulted in 300 g of wet mussel127tissue which was homogenized in a blender. Extraction of the arsenic compounds presentwas carried out by using methanol (2.5 mL per g of wet tissue). The flask containing thetissue-methanol mixture was sealed and placed on a mechanical shaker that was left tooperate for two days. Following completion of the extraction and filtering, the arseniccontent of the extract was determined by using HGAAS and ETAAS.5.2.3.2 Purification Procedures for Arsenic Compounds Present in Mussel ExtractsAn outline of the procedures used for the purification of the arsenic compoundspresent in mussel extracts is shown in Scheme 5.1. The methanol extract, obtained asdescribed above, was evaporated to dryness and the residue was dissolved in water andextracted with portions of diethyl ether until the ether portion was colourless. The waterfraction was reduced to a volume of 15 mL and then applied to a gel-permeation column(Sephadex LH-20, 23 mm I.D. x 415 mm length), and eluted with water. Fractions of 7 mLwere collected, and the arsenic content in 20 jiL aliquots of each fraction were determinedby using ETAAS. Each fraction was analyzed in triplicate, so a total of 60 .tL of solutionwas required. The arsenic containing fractions were combined and evaporated down to avolume of 10 mL and then applied to a strong cation exchange column [Dowex50Wx8(Hj, 200-400 mesh, 25 mm I.D. x 300 mm length]. [The packing material in thiscolumn was initially extracted with acetone, until the acetone became colourless. Theregeneration of the cation exchanger was accomplished by washing it with de-ionized water(500 mL), 2M sodium hydroxide (300 mL), water (500 mL), 2M HCI (300 mL), and finallywith water until neutral]. A step-wise gradient elution procedure was used to elute thearsenic containing compounds. This involved the successive use of the following mobilephases: 120 mL water, 120 mL 5% ammonium hydroxide, 50 mL water and 200 mL 2MHCI. Fractions of 7 mL were collected and analyzed for arsenic. The arsenic containingfractions were combined into three groups, each of which corresponded to a peak eluting128off the Dowex 5OWx8(Hj column with water, ammonium hydroxide and HC1,respectively.Scheme 5.1 Outline of the procedures used for the extraction and purification ofarsenic compounds from mussels.[MolluskJ MeOH extract.1Ether/water partitiomng 4— Dissolve in water,11r___________________Aqueous phase (Gel-permeation[chromatographyStrong Cation exchange Resinwater ammoniurn hydrochloric5.2.3.3 Isolation of Arsenic Compounds in the NH4O FractionThe arsenic containing fractions, that were eluted from the strong cation exchangecolumn by using ammonium hydroxide, were combined and subsequently concentrated onthe rotary evaporator. The solid obtained was dissolved in a minimum amount of water andsubjected to gel-permeation chromatography [GPC] (Sephadex LH-20, 23 mm I.D. x 300mm length) with water as the eluent. Fractions of 7 mL were collected and analyzed forarsenic. The arsenic containing fractions were combined, evaporated to dryness and re129dissolved in a minimum amount of water. This material was then placed on a Dowex2x8(OHj column (15 mm I.D. x 400 mm length), and eluted with 120 mL of water,followed by 200 mL of 0.2 M Rd. Fractions of 10 mL were collected and analyzed forarsenic. The evaporation of the combined arsenic containing fractions resulted in a residuewhich was re-dissolved in a minimum amount of water. Aliquots of this solution wereexamined by using thin layer chromatography (TLC) and HPLC - ETAAS. TLC was carriedout on silica-gel plates, using 65% ethanol - 1% acetic acid - 34% water, as the mobilephase. The Rf values of the arsenic compounds in the extract material as well as the Rfvalues of the standards AsB, AsC, TMA5+1 were determined by scraping 5 mm wide silica-gel strips off the TLC plate, extracting the silica powder obtained from each stripindividually with 5 mL methanol and finally analyzing each extract for its arsenic content byusing ETAAS.The remaining solution was also purified by TLC. Final purification was achieved onan HPLC Protein-Pak DEAE column (7.5 mm I.D. x 75 mm length) with 5 mM ammoniumacetate solution, pH 6.65. A 1 mL.min4 flow rate was used and 0.5 mL fractions werecollected. The arsenic containing fractions produced a residue that was further analyzed bylow and high resolution DCI-MS.5.2.3.4 Isolation of Arsenic Compounds in the HCI FractionThe arsenic containing fractions eluting off the strong cation exchange column byusing hydrochloric acid were combined and then concentrated on a rotary evaporator. Theresidue was dissolved in 0.2 M ammonium hydroxide (15 mL) and then chromatographed byusing GPC (Sephadex LH-20, 25 mm I]) x 350 mm length). Fractions of 10 mL werecollected and analyzed for their arsenic content. The arsenic containing fractions werecombined and evaporated down to 10 mL. TLC was then used as described earlier, both foridentification and purification purposes. Final purification was accomplished on a Protein130Pak DEAE HPLC column. The purified solid was analyzed by using both low and highresolution DCI-MS.5.2.3.5 Determination of Arsenic in Mussel ShellsShells from Californian mussels were divided into two groups. The shells in bothgroups were washed with deionized water, air dried, crushed and ground into a fine powderin a mortar. However, the shells from one of the groups had their outer surface sand-blastedprior to being ground into a fine powder. This procedure was employed in order to removeany organic matter from the outer surface of the shell. The powder (10.0 g) was placed in a250 mL beaker and 40 mL concentrated HC1 was added to dissolve it. After dilution withwater the undissolved material was filtered out. The resulting solution was analyzed forarsenic by using hydride generation -gas chromatography (HG-GC) - AAS.5.3 RESULTS AND DISCUSSION5.3.1 Purification and Isolation of Arsenic Compounds Extracted from McalfornianusThe extraction and separation procedures used, as described above, were based onwork previously published.28’2431,234.Soft tissue from Californian mussels was extracted with methanol. The weight andarsenic content of the mussel tissue and extracts are given in Table 5.1. From this table itcan be seen that 83% of the arsenic present in the mussel’s soft tissue was extracted into themethanol. After evaporation of the methanol the gum like material which remained wasdissolved in water and then extracted with diethyl ether, which removed approximately 4%of the arsenic present in the water solution. This percentage is an indication of the amount131of lipid-like arsenicals present in the original methanol extract. We did not carry out anyfurther work on the ether extracts. The percentage of ether soluble arsenic determined to bepresent in the methanol extract of these mussels, is comparable to those found by others inclam tissue,235 Western rock lobster,236 and shell fish.237 However, higher amounts havebeen reported for crabs (17%),190,238 and brown alga Undaria pinnatIda (25%).34 Anearlier worker suggested that arsenolipids from algae are unstable and are readilyhydrolyzed to water-soluble arsenic compounds.239 Recently however, the arsenicalpresent in the chloroform extract of Undaria pinnat/Ida was isolated by using solventpartitioning, GPC, and HPLC on silica gel, and was shown to be an arsenic containingphospholipid whose structure is shown in Figure 5•334 Evidence for this structuralassignment was provided by its ‘H NIVIR spectrum, and by ‘H NMR and GC-mass spectralanalysis of its hydrolysis products. It was reported that this compound is labile, readilydecomposing during extraction, so its identification is considered a major accomplishment.Table 5.1 Weight and arsenic contents of extracted soft tissue, methanol extract, andresidue.CalifornianmusselsWeight of soft tissue extracted (g) 100Arsenic content of soft tissue (ig) 236Residue after methanol extraction (g) 15Arsenic content of residue (pg) 43Arsenic content of methanol extract (ig) 196Total arsenic extracted (%) 83132Following the ether extraction the water soluble arsenic compounds were subjected toGPC. The arsenic containing material eluted from the Sephadex LH-20 between 70 and 130mL (Figure 5.4). A synthetic standard of AsB eluted within the same elution volume. This isan indication that the water soluble arsenicals present in mussels are not strongly associatedwith other larger molecules in the extracts.The arsenic containing fractions collected off the gel permeation column werecombined, reduced down in volume and then chromatographed on a strong cation exchangeresin. This type of resin has been commonly employed for preliminary separation ofarsenicals of marine origin.231 It should be noted that this chromatography separates thearsenicals into “H20”, “NH4OH”, and “HCl” fractions. The arsenic content of the collectedfractions eluting off the column were determined by using ETAAS, and the resultingchromatogram is presented in Figure 5.5. Of the total arsenic eluting off the strong cationexchange column, 36% was present in the ammonium hydroxide fractions, 60% in thehydrochloric fractions, and 4% in the water fractions. Similar results have been reported for avariety of clam species.231 Synthetic arsenic standards were also chromatographed on thiscolumn. Methylarsonic acid elutes from the column with water, whilst DMAsA and MBelute with ammonium hydroxide. AsC and TMAs elute with 2M Rd.Mi (CH2)nMeFigure 5.3 Structure of an arsenic containing phospholipid present in brown alga.OH(CH2)Me133ci)0Ca-I—0(1)-o:5aciC)Ca-O0C’,.00 14 28 42 56 70 84 98 112 126 140 154 168 182 196 2100.300.250.200.150.100.050.00Retention Volume (mL)Figure 5.4 Sephadex LH-20 - ETAA chromatogram; mobile phase: water; 7 mL fractionscollected; 20 jiL of each injected into the ETAA spectrometer; each fraction analyzed intriplicate.0.120.100.0s0.060.040.020.00Retention Volume (mL)Figure 5.5 Dowex 50Wx8(H) - ETAA chromatogram; mobile phase: 120 mL water, 120mL 5% ammonium hydroxide, 50 mL water and 200 mL 2M HC1; 7 mL fractions werecollected; 20 iiL of each fraction was injected into the ETAA spectrometer; each fractionanalyzed in triplicate.0 35 70 105 140 175 210 245 280 315 350 385 420 4551345.3.2 Identification of an Arsenic Compound Present in the Ammonium HydroxideFractionsThe arsenic containing fractions which eluted off the strong cation exchange columnwere combined, evaporated to dryness, redissolved in water and then subjected to GPC.Only fractions with retention volumes in the range of 118 to 140 mL were found to containarsenic. AsB, a compound which is recovered from the ammonium hydroxide fraction,eluted from the gel-permeation column with a similar retention volume.For further purification the arsenic containing material was subjected tochromatography on a Dowex 2x8 (OH-) column. This yielded a single set of arseniccontaining fractions which eluted from the column with water.TLC was also employed to further isolate the arsenic containing compound. Methanolextraction of the arsenic-containing TLC spot and subsequent evaporation of the solvent,yielded a residue. This residue was dissolved in water and chromatographed on an HPLCProtein-Pak DEAE column with 5 mM ammonium acetate buffer as the eluent. Both ofthese chromatographies not only further purified the arsenic containing material but alsoprovided information concerning the compound’s structure. This was accomplished bycomparing Rf values and retention times resulting from TLC and HPLC, respectively. OnTLC the arsenic containing material gave an Rf value of 0.53, while synthetic MB, whichwas also subjected to the same chromatographic procedure under the same conditions,gave an Rf of 0.53. Other synthetic organoarsenicals have the following Rf values: AsC,0.30 and TMAsI, 0.39. On the Protein-Pak column the arsenic containing material eluteswith the same retention time, 2.5 - 3.5 mm, as synthetic AsB (Figure 5.6).In order to further confirm the structure of the arsenical as being that of MB, massspectrometry was used, in particular the DCI-MS technique developed in Chapter 2. TheDCI mass spectrum (Figure 5.7) obtained by using ammonia as the reagent gas indicatesions at m!z 179 [M+H], 135 [(CH)4As], 120 [(CH3)AsJ, and 105 [(CH3)2As], among135other ions. This mass spectrum corresponds very well to the DCI mass spectrum obtainedfor synthetic AsB, shown in Figure 2.6.The identification of the arsenical was confirmed by using high resolution accuratemass measurements of positive ions in DCI mode. The development of this method has beendescribed in detail in Chapter 2, in particular the procedure followed the analysis asdescribed in Section 2.2.3.2. The accurate mass measurements showed that the m/z 179 ioncorresponds to the protonated molecular ion of AsB. The absolute error, [(Theoreticalvalue-Measured)/Theoretical] x 1 o6, the measured mlz value was calculated to be 3.9 ppm.From these data it can be concluded that AsB is a major component in the Californianmussel. This result reinforces the notion of the virtual ubiquity of AsB in marineinvertebrates and fish. 1,130.12Cl)00.048:80.060.030.000 2 4 6 8 10 12 14 16 18Retention Time (mm.)Figure 5.6 BPLC-ETAAS chromatogram of: A. Synthetic arsenic standards: MB, As(llI),and DMAsA 20 .tL samples containing 500 ng of each arsenical were injected onto .thecolumn, B. arsenic containing material collected off the strong cation exchange column inthe ammonium hydroxide fractions. Chromatographic conditions: Waters Protein PakDEAE column; 5 mM ammonium acetate mobile phase; flow rate 1 mL.min; fractionscollected every 30 s and 20 iiL of each one injected into the graphite tube for thedetermination of arsenic.136LETsuopijpxo1p1(qwniuowuminrnsiuinpxuoiwouoiisjjoprnjjo‘umunnmuoousijowtuidsssw(siuainiourni)IDULS‘!IRelativeIntensityNxxxI.-’__NI.NNNNNN5.3.3 Identification of an Arsenic Compound in the Hydrochloric Acid FractionsThe arsenic containing material recovered from the strong cation exchange resin whenusing HC1 as the eluent, was further subjected to GPC. Arsenic containing fractions elutedfrom this column between the volumes of 118 to 154 mL. This elution pattern is similar tothe one observed for the arsenical which we identified as AsB, present in the ammoniumhydroxide fractions. This indicates that the arsenic containing compound present in the HC1fraction is similar in size to AsB.Further purification of this material was accomplished by using preparative TLC andHPLC.As mentioned earlier TLC and HPLC methods are capable of providing somestructural information regarding the arsenical under investigation. In this case TLC wasused for such purposes. The Rf of the arsenic containing material was determined to be0.38, which is in agreement with that obtained for synthetic TMAs+I.To further confirm that the arsenic compound present in the HC1 fractions is theTMAs ion, MS was employed. Again in this case DCI-MS proved to be invaluable. Thelow resolution DCI mass spectrum is shown in Figure 5.8; with characteristic ions at mlz135, 121, and 105, corresponding to [(CH3)4Asf, [(CH3)As+Hf, and [(CH3)2Asf’,respectively. The DCI mass spectrum obtained is similar to the one reported for syntheticTMAsI (Figure 2.7). High resolution DCI measurements of the m/z 135 ion showed thatit corresponded to [(CH3)4Asf, with an absolute error of 2.2 ppm from the theoreticalvalue.From these results we can conclude that the arsenic compound present in the residueobtained from the HCI fractions is a tetramethylarsonium salt. The presence of such salts inmarine animals (clams: Meretrix lusoria), was first reported by Shiomi et al. 28 Since thenothers have reconfirmed their presence by identi1ying the TMA5+ in a variety of marineanimals.231 Even though the role of the compound is still unclear, it has been proposed that138it is formed via the methylation of Irimethylarsine, with SAM being the likely CH3donor.1’3_________________________________________NNL)_________________—NLr,___‘-4—=‘-4Ltx xLr Lt N£suuj Aj)JFigure 5.8 DCI (ammonia reagent gas) mass spectrum of arsenic containing material,collected off a strong cation exchange resin in the hydrochloric acid fractions.1395.3.4 Attempts to Identify an Arsenic Compound in the Water FractionsAttempts were also made to identi1’ the arsenic containing compound(s) present inthe water fractions. Purification by using GPC, TLC, and HPLC yielded residues for whichno structural assignments could be made based on the DCI mass spectra obtained. Theresidue appeared to consist of a mixture of compounds and so fhrther purification andisolation seemed necessary. TLC showed the presence of only one arsenic containing spotwith an Rf of 0.83. Unfortunately fI.irther investigation of this material was not possiblebecause of its scarcity and/or lack of stability. No previous study has reported the successfulidentification of an arsenic compound that eluted in the water fraction, perhaps for similarreasons.It has been recently reported that arsenosugars are present in bivalves in addition toTAsh50J84Ion-pair reverse-phase HPLC-ICP-MS was used for the identification of thearsenosugars. The advantage in using such a method is the elimination of a number ofsample preparation steps, such as the use of the strong cation exchange resin, which couldchange the original structure of the arsenicals or even introduce artifacts. We suggest that,although it is likely that the water fractions from M cabjornianus contain arsenoribosederivatives, these compounds may not nccessarily be those originally extracted from themussel flesh, because such derivatives are sensitive to extremes of pH conditions and maynot survive passage down the strong cation exchange column. In support of this suggestion,some recent work shows that arsenosugars are eluted partially decomposed from a strongcation exchange resin in the water fractions.24°5.3.5 Arsenic Speciation in Mussel ShellsA HG-GC-AA system was used to separate and detect the arsenic species present inmussel shells which form arsines upon reduction with sodium borohydride. As described inthe Experimental Section, the shells used for this study were divided into two groups, one140contained shells which had the outside part sand blasted and the other remainded untreated.However, once analyzed no pronounced difference was detected in the arsenic content andarsenic species present in these two groups of mussel shells. The shells were found tocontain: 105 ng.g inorganic arsenic [As(V)+As(ffl)], 2.1 ng.g’ MeAs(V), and 10.3ng.g Me2As(V). These results are reliable to ± 5%. A typical chromatogram of thearsines is presented in Figure 5.9. Similar arsenic species have been detected in Blue musselshells, as well as a variety of clam shells.2418Figure 5.9 HG-GC-AAS chromatogram of arsenic containing species, originating fromCalifornian mussel shells.Retention Time (mm.)1415.4 SUMMARYThe results reported in this Chapter establish the presence of AsB and atetramethylarsonium salt in Californian mussels. Initially the arsenic containing materialextracted from mussels was purified and isolated by using a combination ofchromatographic techniques, such as, TLC, GPC, and [{PLC. Comparison of Rf values,retention volumes, and retention times, of the arsenicals present in the mussel extracts withthose of synthetic arsenicals, provided preliminary information regarding their structuralidentity. Finally the purified residues were analyzed by using MS techniques developed inthis work (described in detail in Chapter 2). The use of both low and high resolution DCIMS, allowed for the identification of the arsenicals mentioned.Questions still remain about the identity of the arsenic compound(s) elutingunabsorbed off the strong cation exchange column, even though it is highly possible thatthese compounds are arsenosugars.The presence of methylated arsenic species in the mussel shells was also established.This was accomplished by using a HG-GC-AAS system and identifying the arsines bycomparing their retention times with those of synthetic standards.142CHAPTER 6BIOTRANSFORMANTION OF[311]-METI{YLARSONIC ACID II%i A STATICSEAWATER SYSTEM CONTAINING MYTILUS CALIFOR[VIANUS6.1 INTRODUCTION6.1.1 Scope and Rationale of WorkThe main objective of this study was to investigate the biotransformation of an arseniccompound in the marine environment. More specifically, this experiment was designed tostudy the biotransformation of3H-labeled methylarsonic acid ([3H]-MAsA) in a staticseawater system containing Mytilus cahfornianus, and to identify the [3Hj-MAsAmetabolites accumulated within the mussel flesh. The experiment was conducted in order toprovide answers to a number of questions concerning the arsenic cycle in the marineenvironment, such as: which marine organisms are responsible for the arsenicbiotransformations, what is the identity of the arsenicals produced along this cycle, and howdo these arsenicals interconvert?MAsA was selected as the compound to investigate because it is believed to be aprecursor to the more complex arsenicals found in marine organisms, e.g. AsB, arsenosugars,etc.,44 although a direct link has not been established. Another reason for using MAsA is thatit is one of the least complex arsenicals present in the marine environment and can easily belabeled with tritium.The main purpose for using the radioactive label is to simplify the detection of MAsAmetabolites, even if they occur at very low levels, and also to allow for a simple way ofdifferentiating between the metabolites produced from naturally occurring arsenicals and143those from[3H1-MAsA. Tritium was selected, as opposed to other radionuclides, because itis inexpensive and can be detected at very low levels, it is relatively long lived, and it caneasily be handled with minimum precautions. Other radionuclides, e.g. 74As and 14C, whichcould have been used as labels, do not possess most of these advantages.Mytilus calfornianus mussels collected off the B.C. coast were used because of theirknown arsenic species content (Chapter 5 of this Thesis), and also because they can bemaintained relatively easily compared to the smaller M edulis species.6.1.2 Methylarsonic Acid in the Marine EnvironmentMethylarsonic acid and its derivatives have been reported to exist in various parts of themarine environment; seawater, marine organisms, and sediments.1 The anthropogenic inputof MAsA is believed to be relatively small, except for localized, often accidental discharges,and in spite of the fact that sodium methylarsonate is widely used as a herbicide.5’242-5The presence of methylarsenic species in the marine environment is a consequence of themethylation of inorganic arsenic. It has been demonstrated that this methylation occurs viabiomethylation, i.e., it involves living organisms, rather than just an environmentalmethylation involving a chance abiotic reaction between an available metal/metalloid and abiologically formed methylating agent outside the cell.246The presence of MAsA in seawater is well documented as the result of the work ofAndreae247’8 and others24 on the concentration of MAsA and other arsenic species inthe northeast Pacific and Californian coastal waters. The profiles of arsenic speciesconcentrations versus depth show that the arsenate concentration in the photosyntheticallyactive surface waters (euphotic zone) is decreased, with a corresponding increase in arsenite,MAsA and DMAsA concentrations. In deeper waters the concentrations of themethylarsenicals are at or below detection limits (0.002 ppb). This observation suggests thatDMAsA and MAsA are produced by phytoplankton or closely related heterotrophs. This is144further supported by correlations which were found between typical indicators of primaryproductivity, i.e., chlorophyll concentrations and/or 14C uptake, and depth profiles of theconcentrations of arsenic species. Similar results were reached by Reimer and Thompsonwho studied the distribution of arsenic species present in the British Columbia coastal watersduring a bloom. The methylarsonate profile they reported also indicated that this compoundis produced in the surface water.249Methylarsonic acid has also been shown to be present, at very low levels, in interstitialpore water of both natural marine sediments and sediments contaminated with mine-tailings.Laboratory studies have shown that microorganisms present in these sediments are capableof methylating arsenic under both aerobic and anaerobic conditions.61By using HG-GC-AAS it has been shown that bivalve shells contain methylarsenicals,probably salts of MAsA and DMAsA.241 Because of the method of detection, thepossibility that these are methylarsenic(III) species cannot be completely ruled out. Tracesof MAsA have been reported to exist in dogfish tissue.48 However, the identification ofMAsA was based only on a comparison of the retention times obtained by using HPLC-ICPMS, for both standard arsenicals and those present in the tissue extracts.6.1.3 Arsenic, accumulation and biotransformation experimentsTo date a limited number of accumulation and biotransformation experiments haveprovided results that have contributed to our knowledge of arsenic cycling in the marineenvironment. Arsenocholine (AsC) was converted into arsenobetaine (AsB), glyceryiphosphorylarsenocholine (Figure 6. 1A), and phosphatidylarsenocholine (Figure 6. 1B) in yelloweye mullet following oral administration.250Accumulation experiments with mussels (Mytilusedulis) suggested that AsB is readily taken up from seawater by these animals.229The effects of certain environmental (concentration, temperature, salinity) and biological(tissue parts) variables on 74As-arsenate accumulation and elimination processes in the145mussel Mytilus gaioprovincialis have been studied.251 In this study it was found that arsenicuptake increased with increasing arsenic concentration in the water. The accumulation waspartially supressed at higher external arsenic concentrations. The highest 74As concentrationswere recorded in the byssal threads and the digestive gland. It was also shown that increasedwater temperatures and decreased salinity enhanced arsenic uptake, and that accumulation inthe byssal threads contributed to the elimination of the element from the mussels.In a similar experiment Kiump examined the factors influencing the process of arsenicaccumulation and elimination in a food chain consisting of Fucus spiralis (macro algae)Littoria littoralis (marine snail) Nucella lapillus (predatory marine snail).252 The mostinteresting results obtained from this work which involved 74As-labels were the following:diet is the major source of arsenic for L. littoralis and N lapillus, and arsenic is not biomagnified along the food chain.In an experiment similar to the one reported below, water-soluble3H-labeled arseniccompounds were extracted with phenol from mussels (Mytilus edulis) and seawater afterexposure to[3HJ-MAsA and[3H]-dimethylarsinate.253[3HJ-AsB was found in both themussels and the seawater. The results of this experiment indicate that AsB is biosynthesizedby microscopic organisms, probably primary producers, in the seawater and that it isbioaccumulated quite rapidly by mussels.0+___IIMe3 2-O-P-O-CHI I I0 ORORA) R=HB) R=CO(CH2) H3Figure 6.1 Structures of glycerylphosphorylarsenocholine (6. 1A), and phosphatidylarsenocholine (6.113). Both of these compounds were present in yellow-eye mulletfollowing oral administration of arsenocholine.1466.2 EXPERIMENTAL6.2.1 Instrumentation6.2.1.1 Liquid Scintillation CountingA liquid scintillation counter (Packard Tri.carb® 1900 TR) was used to measure theactivity of 3H-labeled compounds present in the various mussel flesh extracts andchromatographic fractions. The counter was standardized daily by using a standard (PackardStandard #22) which contained 0.2 .tCi of [II1. The scintillation counter was connected toan Epson LX-810 printer.Plastic scintillation vials capable of holding 6 mL of liquid were used throughout thiswork. Each sample was counted in triplicate, for 4 minutes each time. The results obtainedfrom the counts were averaged.6.2.1.2 High Performance Liquid ChromatographyThe HPLC system consisted of a Waters M45 pump, a Waters U6K injector, theappropriate column and an automated fraction collector. Separations were achieved by usingtwo different columns, a Protein Pak DEAE 5PW column (7.5 mm I.D. x 75 mm length,Waters), and a Bondclone 10 C18 column (3.9 mm I.D. x 300 mm length, Phenomenex).6.2.1.3 Atomic Absorption SpectrometryA Varian Techtron Model AA 1275 Atomic Absorption Spectrometer equipped with aVarian GTA-95 accessory, was used to determine arsenic by using Electrothermal AtomicAbsorption Spectrometry (ETAAS). For these determinations a chemical modifier consistingof 500 ppm Pd in 2% citric acid was used. Chapter 3 of this Thesis provides detailsconcerning the method used, as well as the conditions used for the ETAAS analysis ofarsenic.1476.2.2 ChemicalsAsB,104 AsC,’°5TMAs,74and[3H]-MAsA254were synthesized by using literaturemethods, all other chemicals were commercially available. A sample ofglyceiylphosphorylarsenocholine (0.7 mg) was kindly supplied by Dr. K.A.Francesconi.2296.2.3 Collection and Storage of Mytilus cal(fornwnusMussels (Mytilus californianus) collected from Quatsino Sound, B.C., were stored inholding tanks at the University of British Columbia I Federal Dept. of Fisheries and Oceans,West Vancouver Laboratory, for 4 months prior to the experiment. These facilities arecapable of providing a continuous flow of seawater which is subsequently aerated in theholding tank (Figure 6.2).A)Figure 6.2 Seawater tanks used for mussel storage and exposure experiment: A: 200 Lseawater tank with continually flowing seawater, used for mussel storage; B: 15 L experimenttank containing mussels and{3H]-MAsA; *: aeration lines for both A and B.Seawater from ocean* B) *Mussels I7Seawater into ocean1486.2.4 Procedures for the Speciation of3H-Labeled Compounds Extracted FromMytilus calfornianusFor the experiment 21 mussels were selected randomly and were placed in a static butwell aerated tank (Figure 6.2B) containing 15 L of seawater and 34 jiCi[3H]-MAsA (1.5ppm As). The mussel shells varied in length from 7 to 16 cm.After 9 days, 9 mussels were removed and shucked, providing a total of 200 g of wettissue. The tissue was homogenized, 500 mL of methanol was added, and the mixture wasthen placed on a shaker for 2 days. This extraction step was repeated with another 500 mL ofmethanol. The extracts were combined and evaporated to dryness and the residue wasdissolved in water and extracted with portions of diethyl ether until the ether portion wascolorless. The water fraction was evaporated down to a volume of 25 mL, applied to a gelpermeation column (Sephadex LH-20, 25 mm ID. x 300 mm length), and eluted with water.Fractions of 9.5 mL were collected, and the 3H activity in each was determined bywithdrawing a 500 1iL aliquot which was mixed with 5 mL of scintillation liquid in a countingvial, before being transferred to a scintillation counter where disintegrations per minute(DPM) were measured. The 3H containing fractions were combined and evaporated down toa volume of 10 mL and then applied to a strong cation exchange column [Dowex 50Wx8(Hj, 200-400 mesh, 25 mm I.D. X 300 mm long columnj. The following mobile phases wereused to elute the 3H containing compounds: 200 mL water, 200 mL 5% ammoniumhydroxide, 40 mL water, and 150 mL 2M HC1. Fractions of 9.5 mL were collected andanalyzed for 3H activity. The 3H containing fractions were combined into four main fractions,each of which corresponded to a peak eluting off the Dowex 50Wx8 (Hj column. Thesefractions were then evaporated to dryness and redissolved in a minimum volume of water.Each of these four solutions was chromatographed on an anion exchange Protein PakDEAE column. Two mobile phases were used, the first consisted of 5 mM sodium acetateadjusted to pH 4 with acetic acid and the second consisted of 5 mM ammonium acetate, pH1496.8. A Bondclone 10 C18 reversed-phase column was also used for ion-pair reversed-phaseliquid chromatography, with water-methanol 95:5 as the mobile phase and 5 mMtetrabutylammonium nitrate as the ion pair. The flow rate in all cases was 1 mL.min4.Inorder to establish retention times for identification purposes a number of standard arsenicals[AsB, methylarsonic acid (MAsA), dimethylarsinic acid (DMAsA), glycerylarsenocholine(GPAC)] were also chromatographed. The standards were detected by using ETAAS: 15 .tLaliquots from 0.5 mL fractions. The sample extracts were monitored by counting aliquots of 1mL that were mixed with 5 mL of scintillation liquid.6.2.5 Determination of Total H In Mussel Tissue and ShellsOn each of the third, sixth and ninth days, 4 mussels (M calfornianus) were removedfrom the tank and dissected into six parts: gills, adductor muscle, foot, mantle, muscle tissueand visceral mass. The same tissue parts from each of the four mussels were combined. Thebyssal threads and the shells were also set aside for total 3H determinations. All of the tissueparts were freeze dried and then ground into a fine powder.The Oxygen Flask combustion method was used to prepare the mussel tissue for 3Hliquid scintillation counting. This method has been used in a wide range of analyticalapplications as well as for the determination of 3H and 14C activities in biologicalsamples.255’6The apparatus consists of a 3L round-bottom flask, a stopper and a platinum basket(Figure 6.3). The freeze dried sample (100 mg) was wrapped in a paper sample wrapper andplaced into the platinum basket. The flask was filled with oxygen and then the tip of thesample wrapper was ignited and the stopper and basket were inserted into the flask. Oncecombustion had ceased 5 mL of absolute ethanol was added to absorb the[3H]-H20produced. A 2 mL aliquot of the alcohol solution was transferred to a counting vial and 4 mL150of scintillation liquid was added prior to counting. This procedure was performed in triplicatefor each tissue sample.Shells were washed with deionized water, air dried, crushed and ground to a fine powderin a mortar. The powder (2.0 g) was placed in a 250 mL beaker and 10 mL of 2M HC1 wasadded. After dissolution the solution was filtered to remove the insoluble residue. 500 1iLportions were withdrawn and mixed with 5 mL of scintillation liquid and then counted.AignitionsampleFigure 6.3 Oxygen Combustion Flask (3L); A. platinum basket, B. paper sample wrapper, C.pressure relief in the form of a rubber balloon.1516.3 RESULTS AND DISCUSSION6.3.1 Speciation of the3H-Labeled Compounds Extracted From M. californianusMussels were exposed for 9 days to seawater containing[3Hj-MAsA. The pitJiininaiymethanol treatment of their tissue extracted 74% of the 3H-labeled compounds. Theextraction efficiency was determined by measuring the 3H activity of the mussel flesh beforeand after the extraction. The methanol extract was evaporated to dryness and redissolved inwater. Diethyl ether extracted approximately 9% of the counts from this solution which isprobably a measure of the amount of lipid-like arsenicals in the original methanol extract.The water soluble3H-labeled compounds eluted from Sephadex LH-20 between 100 and140 mL (Figure 6.4). Standard AsB eluted within the same retention volume. This is anindication that the3H-label has been incorporated into water soluble compounds that exhibitsimilar physical properties (size and/or adsorption characteristics) to those of AsB, on thisparticular column.1 4001200100080060040020000 2 4 6 8 1012141618202224262830Fraction NumberFigure 6.4 Sephadex LH-20 liquid scmtillation chromatogram; water mobile phase; 9.5 mLfractions collected; 0.5 mL of each fraction mixed with 5 mL scintillate and counted.152<—H20————><———NH4H — > <———HCI———>90080070060004003002001 000Fraction NumberFigure 6.5 Dowex 50Wx8 (Hj liquid scintillation chromatogram; mobile phase 200 mLwater, 200 mL 5% ammonium hydroxide, 40 mL water and 150 mL 2M HC1; 9.5 mLfractions collected; 0.5 mL of each fraction mixed with 5 mL scintillate and counted.04812162024283236404448525660153A strong cation exchange resin has been commonly employed to accomplish apreliminary separation of the arsenicals in marine animals into water, ammonium hydroxideand hydrochloric acid eluting fractions.28’2431This procedure was also used in thepresent study. The radioactivity in the fractions eluting from the Dowex 5OWx8 (Hj wasmeasured and the resulting chromatogram is presented in Figure 6.5. Four peaks containing3H-labelecl compounds eluted from this column. The first peak contained a compound (thiscompound will be referred to as PEAK #1) that essentially showed no interaction with thestrong cation exchange resin. The compounds comprising the second (Peak #2) and third(Peak #3) peak were weakly retained by the resin, and were eluted by using water. Finally thecompound in the fourth peak (Peak #4) was eluted by a 5% ammonium hydroxide solution.Methylarsonic acid elutes from this type of column with water, whilst DMAsA and AsB elutewith ammonium hydroxide. AsC and TMAs elute with 2M HC1. No 3H activity wasdetected in any of the fractions when 2M HC1 was used as the eluent for the mussel extracts.After obtaining this preliminary information about the properties of the 3H-labeledcompounds, HPLC was used for further identification.6.3.1.1 Identification of[3H]-MAsA and[3H]-AsB in mussel extractsThe HPLC Protein Pak DEAE chromatograms (5 mM sodium acetate, pH 4, mobilephase), of Peaks #3 and #4, and of a mixture of standard AsB, DMAsA and MAsA arepresented in Figure 6.6. Standard MAsA and Peak #3 exhibit the same retention time. Peak#4 also has the same retention time as standard AsB and DMAsA, which are not separatedunder these conditions. However, these two standards are readily separated on the ProteinPak column by using 5 mM ammonium acetate (pH 6.8) as the mobile phase.74 This ispresented in Figure 6.7, along with the elution profile of Peak #4. Peak #4 exhibits the sameretention time as standard AsB.15410 20 25 30o 0.5< 0.4- 0.30a)0.1U)0 0.0600400a2001501 00a5000Retention Time (mm)Figure 6.6 HPLC conditions for A, B, C: Waters Protein Pak DEAE column; mobile phase 5mM sodium acetate, pH adjusted to 4 with acetic acid; flow rate 1 mLmin; fractionscollected every 1 mm;A. HPLC-ETAA chromatogram of standards a: arsenobetaine (250 ng As), b:dimethylarsinicacid (500 ng As) , c: methylarsonic acid (500 ng As).B. HPLC-liquid scintillation chromatogram of Peak #3; 1 mL fraction mixed with 5 mLscintillate and counted.C. HPLC-liquid scintillation chromatogram of Peak #4; 1 mL fraction mixed with 5 mLscintillate and counted.0 5 155 10 15 20 25 301550 2 4 6 8 10 12 14 16 18 20804016 18 20Figure 6.7 HPLC conditions for A and B: Waters Protein Pak DEAE column; mobile phase5 mM ammonium acetate, pH 6.8; flow rate 1 mLmin.A. HPLC-ETAA chromatogram of standards a: arsenobetaine (250 ng As), b:dimethylarsinicacid (250 ng As); 0.5 mL fractions collected every 0.5 mm.B. HPLC-liquid scintillation chromatogram of Peak #4; 1 mL fraction mixed with 5 mLscintillate and counted.0.5a) 0.4- 0.3a)0-. 0.2Q0U 0000 2 4 6 8 10 12 14Retention time (mm)1560 6 9 12 15aU)L 0.3-aU)0— 0.1U)..D 0.0< 500400300o 2001 001 2090a... 6003000 15Retention Time (mm)Figure 6.8 HPLC conditions for A, B, C: Bondclone 10 C18 reversed phase column; mobilephase water-methanol 95:5; ion-pair reagent 5 mM tetrabutylammonium nitrate; flow rate 1mLmin1.A. HPLC-ETAA chromatogram of standards a: arsenobetaine, b: dimethylarsinic acid, C:methylarsonic acid; fractions collected every 30 sec.B. HPLC-liquid scintillation chromatogram of Peak #3; 1 mL fraction collected and mixedwith 5 mL scintifiate and counted.C. HPLC-liquid scintifiation chromatogram of Peak #4; 1 mL fraction collected and mixedwith 5 mL scintillate and counted.33 6 9 12157Ion-pair reversed-phase liquid chromatography was also used for these identifications.Figure 6.8 presents the elution profile of standard AsB, DMAsA, MAsA and that of Peak #3and #4. Again Peak #4 is identified as AsB and Peak #3 as MAsA.All of these chromatographic results are summarized in Table 1.Table 6.1 Summary of chromatographic experiments.Dowex H HPLC HPLC RPLC C-18(Eluent) Anion Exchange, Anion Exchange, Ion-Pair,5mM sodium 5mM 5mM tetrabutylacetate, pH 4 ammonium ammonium nitrate,acetate, pH 6.8 pH 6.8(tR, mm.) (tR, mm.) (tR, mm.)Compound H20 15.5±0.5 nd 10.75±0.25proposed tobe MAsA(PEAK #3)Standard H20 15.5±0.5 nd 11.0±0.5MAsACompound 5% 3.5±0.5 3.5 ±0.5 5.5±0.5proposed to NH4ObeAsB(PEAK #4jStandard 5% 3.5±0.5 3.25±0.25 5.75±0.25AsB MI4OHStandard 5% 3.5±0.5 14.25±0.25 8.25±0.25DMAsA NH4OPEAK #2 H20 8.0±0.5 3.5 ±0.5 ndnd : not determined158These results are significant in that they indicate that MAsA is a precursor to AsB. Otherexperiments,251,257 of similar nature, have been unsuccessful in demonstrating that a simplearsenic compound, i.e.[74As]-arsenate, is a precursor to AsB. These experiments involvedeither direct feeding of the precursor arsenate to a marine animal or exposing one specificalga to the precursor in the growth medium. In contrast, the present study used naturalseawater, which contains different types of algae, bacteria, phytoplankton, and diatoms, anyone of which, or any combination, could be involved in the biotransformations necessary forthe production of AsB.Only limited information can be gained from this experiment regarding the stage in thefood chain at which the arsenical interconversion takes place, even when other resultsobtained from this work (Chapter 5) and data of other researchers are considered.AsB has been found to occur naturally in Mytilus californianus, while MAsA has notbeen detected (Chapter 5 of this Thesis). Thus the ratio of[3H]-MAsA to[3H]-AsB foundin the mussel flesh following exposure to[3Hj-MAsA does not reflect the natural ratio. Itshould be pointed out, however, that the high arsenic concentration (1.5 ppm As) in thestatic seawater system, may have caused overloading of the mussels thus not allowing themto function in a natural way. Thus we can only speculate that the conversion of[3H]-MAsAinto[3Hj-AsB does not take place within the mussel itself This is based on the fact that if[3H]-MAsA was readily converted to[3H]-A5B within the mussel tissue then the measuredcontent of[3H]-MAsA would be much lower. If however the conversion is a slow processwe would expect to find naturally occurring MAsA within this mussel and this, as indicatedabove, is not the case.These results indicate that[3H]-AsB is either accumulated from water and/or food, ormay be synthesized from arsenic compounds other than MAsA within the mussel itselfCullen and Nelson253 reached similar conclusions from studies with Mytilus edulis, abivalve that readily takes up AsB from seawater.229 Wrench et. al.254 studied a three step159food chain consisting of an autotroph, a grazer and a carnivore. They concluded that themuscle tissue of the carnivorous shrimp could not itself form “organic arsenic”, which isbelieved to be synthesized by primary producers.6.3.1.2 Attempts to identify Peaks #1 and #2Peaks #1 and #2 have also been chromatographed under all the above conditions. Theresulting retention times do not correspond with any of the standard arsenicals available to us.Glyceryiphosphorylarsenocholine (Figure 6. 1A), a compound that has been found toaccumulate in yellow-eye mullet following oral administration of arsenocholine, was alsotested.25°The reported chromatographic behavior of this compound seemed to be similar tothose of Peaks #1 and #2. However, the retention times acquired for this compound on theProtein Pak column did not match those ofPeaks #1 and #2.Thus questions still remain about the identity of these two compounds that were notidentified. Only recently has it been established that arsenosugars are present in bivalves.30’Although it is likely that PEAKS #1 and #2 contain arsenoribose derivatives, these may notnecessarily be those originally extracted from the mussel flesh, since such derivatives arereported to be sensitive to extremes of pH and may not survive passage down the strongcation exchange column.259’4°6.3.2 Determination of Total H in Mussel Tissue and ShellsAfter 3, 6, and 9 days of exposure to[3H]-MAsA containing seawater the specificactivities detected in the M calfornianus mussel tissue were 27, 42, and 65 dpm.mg1respectively. The distribution of 3H activity within the mussel was also determined. After themussels had been exposed for a 9 day period the highest specific activity was found in thevisceral mass and the gills of the mussel (Figure 6.9). Similar distribution of the activity withinthe tissue parts was observed on days 3 and 6.160No 3H activity was detected in the shells after using the sample preparation proceduredescribed, indicating that physical processes such as surface sorption play a minor role in theuptake of arsenic compounds by the mussel shells. Methylarsenicals have been shown to bepresent at very low levels in shells of M cahfornianus (Chapter 5 of this Thesis), M edulisand shells of various clams.241 The absence of 3H activity in the shells we examined,indicates that the incorporation of arsenic compounds in shells is a process that requires morethan 9 days to occur. This would be consistent with the idea that the mainly inorganic arsenicspecies to be incorporated into the shell are produced via biomineralization processes ratherthan just surface sorption. Others have reported that radioactive 74As-arsenate added toseawater is found in (or on) the shells of gastropods. In general, there are few reports in theliterature regarding the presence of arsenicals in mollusk shells.It has been proposed that byssal threads act as sinks for the removal of certainradionuclides and heavy metals from contaminated mussels.260262 This has also been shownto occur with arsenic (as74As-arsenate) elimination from mussels.251 In our work the byssalthreads were found to contain an appreciable amount of 3H activity thus supporting thenotion of arsenic elimination and removal via the byssal threads.16120>..t 15 ±>-IC,10 ++C,IiiLI)0— - — — I I I I4 Ct) I- Lii Lii U)_J 0 - -‘ 0 -‘C) I- 4 Liio ZE Li Cfl c_) U)U) D Cl) Ct)5; 0 >-4Mussel PartsFigure 6.9 Distribution of 3H activity within mussel parts, sampled on day 9.6.4 SUMMARYA great deal of information concerning the arsenic cycle in the marine environment canbe obtained by conducting arsenic accumulation and biotransformation experiments, such asthose presented in this chapter.The major fmdings of this study are the following: when Mytilus californianus isexposed to[3H]-MAsA in a static seawater system,[3H1-MAsA,[3H1-AsB, and twounknown3H-labeled compounds accumulate in the tissue parts of the mussel, also thehighest specific activity is found in the visceral mass and the gills of the mussels.This work establishes that MAsA is a likely precursor to AsB. It also indicates that162strong anaerobic conditions are not required for the formation of AsB, as has previouslybeen proposed.42’229Because experiments of this type can add to our knowledge of arsenic cycling in theenvironment, additional studies should be conducted to address a number of stillunanswered questions, for example:(a) Once suitable arsenic standards become available, the task of identifying the twocompounds, which were not successfully identified in this study (Peaks #1 and #2), shouldbe pursued. The identity of these compounds may allow us to establish the intermediates inthe biosynthesis of arsenobetaine.(b) The biotransformations of other arsenic compounds which exist in the marineenvironment should also be investigated. For example, TMAs+ is a compound whosepresence in marine animals is well established but whose origin and role is completelyunknown. For example, is this compound further metabolized?(c) Also of great interest would be to identify the stage in the food chain at which theinterconversion of the arsenicals occurs. This can only be accomplished by conducting wellcontrolled feeding experiments, involving selected organisms at various trophic levels andinvestigating their capabilities to biotransform arsenic compounds.(d) The relationship between the biomineralization processes responsible for shellformation and the presence of arsenic in the shells should be established. This can beaccomplished through similar experiments of longer time duration. Shell layers would haveto be removed and analyzed separately in order to investigate the presence of3H-labeledarsenic in the newly formed layers.163CHAPTER 7SUMMARYIn the previous chapters of this thesis, research concerning the development andapplication of analytical methods for arsenic speciation was described. During this work, amajor emphasis was placed on the development and evaluation of mass spectrometrictechniques for the analysis of arsenicals found in both the marine and terrestrial environment.In particular, DCI-MS, high resolution DCI-MS, MALDI-TOF-MS, dynamic-LS1MS-MS,thermospray-MS, LSIMS, and HG-GC-MS were investigated.Satisfactory DCI-MS spectra can be obtained with 100 ng arsenic placed on the DCIfilament, making the technique eminently suitable for the investigation of environmentalsamples. The spectra of the arsenicals offered recognizable molecular ions as well ascharacteristic fragment ions.To extend the usefulness of DCI-MS, an analytical method was developed thatpermitted the use of mass deficient reference standards for calibration purposes in accuratemass measurements of positive ions under ammonia DCI conditions. This was accomplishedby employing a mixture of ammonia and methane as reagent gases. In the high resolutionaccurate mass measurement experiment, this gas mixture allows for simultaneous detectionof the mass spectrum of perfiuorokerosene (calibration substance) adequate for calibrationpurposes, and the spectrum of the analyte which contains molecular weight information. Aneedle valve system was used to control the composition of the gas mixture introduced intothe ion source. For positive ion accurate mass measurements of higher masses (up tomlz=23 00), Fomblin 18/8 oil was used successfully as a reference standard under ammonia,methane, and iso-butane desorption chemical ionization conditions.164Both low and high resolution DCI-MS were used to identif,’ arsenic compoundspresent in Mytilus cahfornianus. This was accomplished by first extracting the arsenicalsfrom the mussel flesh with methanol, and then isolating and purifjing the compounds bymeans of conventional chromatography. Subsequently the mass spectrometric techniquesdescribed above were used to analyze the purified materials and provide spectra suitable forthe structural characterization of two principle arsenic species present in the mussels;arsenobetaine and the tetramethylarsonium ion.MALDI-TOF-MS is another mass spectrometric technique which exhibits potential foruse in the speciation of arsenic in the environment. The method is very sensitive and theminimum detectable amount was determined to be 0.3 ng of arsenic or 4 pmole ofcompound. This feature suggests that the method could be used for the identification ofminor arsenic components in environmental samples. Other advantages which make itattractive for arsenic speciation are the following: a) the method is capable of providingmolecular ions as well as fragment ions for a variety of arsenicals, b) compoundfragmentation can be controlled to a certain extent by adjusting the N2 laser power, and c)the analysis requires only very small sample volumes 1-1.5 1.tL.HG-GC-MS was also developed in this work and was used to provide conclusiveevidence of -CD3 incorporation from L-methionine-methyl-d3into arsenic compoundsproduced from arsenate by alga cell cultures. The results from this work are of greatimportance because they contribute towards the elucidation of the mechanism of arsenicbiomethylation. These findings strongly support the notion that the oxidation-reductionpathway involving carbonium ions, as originally suggested by Challenger for the alkylationof arsenic by microorganisms (Figure 1. i),44 applies to marine cellular alga and probablyother marine organisms.The advantages of micro-HPLC were demonstrated by the work reported in Chapter 4of this thesis. Micro-LC columns fabricated and packed in house can be conveniently165coupled on-line to a variety of mass spectrometric systems, mainly because of the extremelylow flow rates they require. It was shown that a 99 % reduction in the volume of solventwaste is achieved by switching from conventional to micro-LC, and a 87.5 % reduction byswitching from microbore to micro-LC. This reduction in solvent waste can translate intolarge savings, in terms of both solvent consumption, and solvent waste handling anddisposal. Compared to previous work, lower detection limits for arsenic compounds werealso obtained when micro-LC columns were employed in conjunction with UV and ETA.Adetectors.74The micro-LC columns are able to efficiently separate arsenicals that are used asanimal feed additives as well as their potential metabolites. This separation technique inconjunction with a variety of compatible detectors, can help in the evaluation of thephysiological role of these compounds as well as to assess their impact on the environment.When considering the general use of these feed additives and their possible impact on theenvironment and public, this method is of prime importance because only a small number ofanalytical techniques had been developed for the analysis of this class of compounds. Mostof these are for target analysis of a few specific compounds, which would probably beunsuitable for the detection of metabolites.In order to accomplish the accurate determination of arsenic in environmental samplesit is essential that the optimization of the analytical method used be performed in conjunctionwith a standard reference material of similar matrix to that of the sample being investigated.In the present work Simplex optimization was used to efficiently delineate the optimumexperimental conditions to be used for the electrothermal atomic absorption spectrometricanalysis of total arsenic in a standard reference material of marine origin. Four experimentalvariables, were considered: ashing temperature, atomization temperature, modifierconcentration, and atomization ramping time. This combination of methods and materialsprovides a powerful means of rapidly optimizing the experimental conditions used for the166analysis of arsenic in a wide variety of samples of environmental origin. Excellent recoveriesof arsenic were obtained when using the optimum electrothermal atomic absorptionspectrometry conditions to analyze standard solutions of arsenobetaine, arsenocholine andtetramethylarsonium iodide. This procedure also allowed for the accurate determination ofarsenic in Californian mussels collected from a variety of locations along the B.C. coast.Finally, in Chapter 6 it was demonstrated that MAsA is a likely precursor to MB inthe marine environment. Mytilus calfornianus exposed for 9 days to a seawater systemcontaining[3H]-methylarsonic acid, was found to contain[3H]-methylarsonic acid alongwith[3H]-arsenobetaine and two unknown3H-labeled compounds in the tissue parts of thismussel. 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Temperature settings: Probe at129 °C, vaporizer at 185 0C, source block at 223 °C, and jet at 120 °C.183V81DoOZJi1iu‘Doipoqamos‘Do8Ji1zuodA‘Do611iqoij:suusppuosiupCuqdxoip-jouin.odssswidsow.iijRelativeintensityIIIIIIIIII.—I°1=118Iif‘Doipoqamos‘jçjiizuodA‘306Z1wqoij:suusaEfl11QdtUj.pporuosipCuqd&xoJpi(4-fr-011!U-EjounudssswidsouuqRelativeIntensity.1__________________+C’++‘1—f C’ZC*I—153(-)LSIMS0 z*+I’306* 168*381z1991 246395*107214289442516—IIittTh1LLr1T-,4FiT—.•100150200250300350400450500M!Z+a.163Cl)(+)LSIMSCDz*Z154+++i*+383518107*34745*502176248214289329___441473___0—rrprir1{1TLIL1i1ii100150200250300350400450500M/ZLSIMSmassspectraof4-nitrophenylarsonicacidinnegativeandpositivedetectionmode.LSTpowuopmpArnSOdpuAflUUtponjiutsi-djouxidsswsiisiRelativeIntensity0III-----II——[M+H]-[M-H]c12—%-0z*r—[M+NBA,-H2O,+H][M+NBA,-H20,-H]w“3[M+2NBA,-2H2O,+H]—[M+2NBA,-2H 0,-H]881uoupAtTsOdpuuipiowosiuqdiCxoip(q-joidsssmRelativeIntensity+2C+2“I-.powswisi+2—‘.I0—I0-—0* .—-E_-[M-HjL[M+H]:*+Cfi-I0C1C17—U’ -[M+2NBA,2H2O,+H1—*100%50%0%MJZMass spectrum of 2,6-dimethyiphenol obtained by using DLI-MS; amount injected 500 ng;mobile phase: 80% methanoll 20% water; flow rate: 2 pL.min1;ion source pressure: 10-1torr; masses scanned: 120-140 amu; temperature of copper sleave 180 °C.128 130 148 158 168 178 188 198190

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