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1 SPECTROSCOPICTECHNIQUESININDUSTRIALHYGIENE Spectroscopic Techniques in 5 Chromatographic Spectrophotometric Detectors 4805 Industrial Hygiene 5.1 Introduction 4805 5.2 Spectrophotometric Detectors for High-performance Liquid Chromatography 4806 JinWang,PaulD.Siegel,DanielM.Lewis,Evanly 5.3 Spectrophotometric Detectors for Vo,andWilliamE.Wallace Gas Chromatography 4806 USDepartmentofHealthandHumanServices, 6 Infrared and Raman Spectroscopies 4807 CentersforDiseaseControlandPrevention, 6.1 Introduction 4807 NationalInstituteforOccupationalSafetyand 6.2 Infrared Spectroscopy 4808 Health,Morgantown,USA 6.3 Raman Spectroscopy 4809 7 Field-portable Spectroscopy 4810 7.1 Introduction 4810 KevinAshleyandLloydE.Stettler 7.2 Portable Gas and Vapor Analyzers 4810 7.3 Portable Aerosol Monitors 4812 USDepartmentofHealthandHumanServices, CentersforDiseaseControlandPrevention, Abbreviations and Acronyms 4814 NationalInstituteforOccupationalSafetyand Related Articles 4815 Health,Cincinnati,USA References 4815 1 Introduction 4784 Over the past few decades the pace of change in spec- 2 Mass Spectrometry 4786 troscopic techniques has been remarkable. Spectroscopic 2.1 Introduction 4786 techniques areemergingas important, powerful, and ver- 2.2 Gas Chromatography/Mass satile tools in determining exposure levels of hazards Spectrometry 4787 generated in working environments. Occupational safety 2.3 Liquid Chromatography/Mass Spec- and health studies employ spectroscopic techniques to trometry, Liquid Chromatography analyzehazardouschemicals,biomarkers,andparticulate Tandem Mass Spectrometry and mattersofexposure.Incomparisonwithmanytraditional Liquid Chromatography Ion Trap detection techniques such as gravimetric methods, spec- Mass Spectrometry 4790 trometric techniques are much more sensitive, selective 2.4 Inductively Coupled Plasma Mass and accurate. The major spectroscopic techniques used Spectrometry 4793 in industrial hygiene include mass spectrometry (MS), 2.5 Time-of-flight Mass Spectrometry, scanning electron microscopy (SEM), X-ray microanal- Fourier Transform Ion Cyclotron ysis (XM), atomic spectrometry (AS), ultraviolet/visible Resonance Mass Spectrometry and (UV/VIS) photometry, fluorescent spectrometry (FS), Future Trends 4795 Fourier transform infrared (FTIR) spectroscopy, and 3 Scanning Electron Microscopy and X-ray Ramanspectroscopy(RS).InterestinusingMSinindus- Microanalysis 4796 trial hygiene is driven by its value in understanding basic 3.1 Introduction 4796 physical, chemical, and biological processes related to 3.2 Sample Preparation 4797 workers’exposure to occupational hazards,and in devis- 3.3 Lung Particle Analysis 4798 ing new methodologies to monitor exposures. SEM has 4 Atomic Spectrometry 4801 becomeparticularlyusefulinthestudyofpneumoconioses 4.1 Introduction 4801 and workplace environmental particles since being com- 4.2 Flame Atomic Absorption plemented with energy dispersive X-ray (EDX) analysis Spectrometry 4801 andautomatedimageanalysiscapabilities.SEMandEDX 4.3 Electrothermal-, Hydride Generation- havebeenusedextensivelytocharacterizeparticlesfound and Cold Vapor Atomic Absorption in lung tissues. Atomic spectrometric methods are used Spectrometry 4802 widely for occupational health evaluation of inorganic 4.4 Inductively Coupled Plasma Atomic metals. The development of inductively coupled plasma Emission Spectrometry 4804 atomic emission spectrometry (ICPAES) techniques has EncyclopediaofAnalyticalChemistry EditedbyRobertA.Meyers.(cid:211) JohnWiley&SonsLtd,Chichester.ISBN0471976709 2 INDUSTRIALHYGIENE become increasingly attractive, and has been applicable ionization (MALDI) have contributed to great strides to analysis of nearly all the elements. FTIR and Raman forward in the study of biological macromolecules. The spectroscopiesareemployedtodetecthighlytoxicgasand ability to ionize polar, labile and involatile species has vapor mixtures. Additionally, field-portable methods for been the fundamental basis upon which MS extends monitoring airborne workplace contaminants and toxins its applications. It is important for us to recognize the have received increasing attention. To date, highly spe- breakthroughs in ionization techniques of the 1980s cific, selective, and sensitive spectroscopic technologies and 1990s. These advances have allowed application haveallowedforthedevelopmentofnovelmethodologies of MS to progress and develop rapidly. Additionally, andnewindicatorsforexposurecharacterization.Assess- combining gas chromatography (GC) or liquid chro- mentofactualbodyburdenofchemicals,whicharemore matography (LC) with MS offers the possibility of directly related to potential adverse occupational health taking advantage of both chromatography as a pow- effects, can be accomplished. The major spectroscopic erful separation technique and MS as a powerful and techniquesandtheirapplicationstoindustrialhygieneare sensitivedetectionandidentificationtechnique.Further- describedinthisarticle. more, Fourier transform ion cyclotron resonance mass spectrometry(FTICRMS),andanewgenerationofhigh performancetime-of-flightmassspectrometry(TOFMS), 1 INTRODUCTION whichincludesnanoflowelectrosprayhybridquadrupole time-of-flightmassspectrometry(QTOFMS)andmatrix- Thepaceofchangeinspectroscopictechniqueshasbeen assisted laser desorption ionization/time-of-flight mass remarkable. They are emerging as important, power- spectrometry (MALDI/TOFMS) technologies, are just ful, and versatile tools, and have increasing applications beginning to add much needed analytical power to our in the workplace. For occupational safety and health arsenalformacromolecularidentificationandcharacteri- studies, spectroscopic techniques are primarily used to zation.Thesedevelopmentswillbringimportantmethod- detectandanalyzehazardouschemicals,biomarkers(e.g. ology to scientists and thus accelerate the integration metabolites,DNAadducts,proteinconjugates,andaller- of MS into work-related research strategies. The range gens),andtoinvestigatepneumoconiosesandworkplace of applications in environmental health and industrial environmental particles. They play an important role in hygiene studies includes development of qualitative and determining exposure levels of hazards generated in the quantitative analyticalprocedures,structuraldetermina- working environment, and in finding the adverse effects tionofaberrantproteins,investigationofbiomarkersfor of exposures and their mechanisms of action. In com- exposuretohazards,characterizationofallergensrelated parison with many traditional detection techniques such to occupational asthma, and identification of microbial as gravimetric methods, spectrometric or spectrophoto- products in complex samples. MS and its related tech- metrictechniquesaremuchmoresensitive,selectiveand niquesandapplicationsaredescribedinsection2. powerful. The major spectroscopic techniques used in SEM is introduced in section3. It is ideally suited industrial hygiene studies include MS, SEM, XM, AS, to study pneumoconioses and workplace environmental UV/VIS photometry, FS, FTIR, and RS. In this article, particleswhenitisequippedwithanEDXanalysissystem the basic principles of these major spectroscopic tech- and an image analyzer (IA). Particle matter has been niques and their applications to industrial hygiene are recognized as a cause of various lung diseases for many described. years. Specific pneumoconioses such as coalworker’s The interest in using MS by scientists in studies per- pneumoconiosis,asbestosis,andsilicosismayresultfrom tinent to industrial hygiene is driven by the need to inhalationofparticlesintheworkplaces.SEMandEDX understand the basic physical, chemical, and biological havebeenusedextensivelytocharacterizeparticlesfound processes related to workers’ exposure to occupational in lung tissues. While it is not possible to compare a hazards and to devise new methodologies to monitor measuredlungdustburdentotheactualexposuredueto exposures in the work environment. MS is capable of the lack of exposure data and to particle clearance, the lookingatthedetailsofexposures,andasthemostsensi- measuredlungparticulateburdendoesrepresentretained tivetool,toanalyzesmallmoleculesandmacromolecules particle dose. The SEM and EDX analysis may be used in biological systems. With tandem mass spectrometry for a number of applications in occupational safety and (MS/MS) techniques, MS brought special capabilities to health including the analysis of particles collected on providing specific characterization of molecular struc- air filter samples taken from the working environment, tures and to detecting target analytes at trace levels. analysisofindividualparticlesfoundinbulkdusts,andin The development of new ionization methods such as thestudyofpneumoconioses. electrospray ionization, atmospheric pressure chemical Bulk compositional analyses of respirable particulate ionization (APCI), and matrix-assisted laser desorption material, and even particle-by-particle compositional 3 SPECTROSCOPICTECHNIQUESININDUSTRIALHYGIENE analyses, may not always be sufficient to predict the liquid chromatography(HPLC)or GCinclude UV/VIS, level of biological activity of respirable particles or the fluorescent,lightscattering,refractiveindex,diodearray, health risks of exposure to them. Toxicants may be MS, infrared (IR), radioactivity, and luminescence. The located on the particle surface or may be more heavily choiceofdetectorisdependentontheanalyte’sspectral concentrated there, resulting in heightened biological properties, and the required sensitivity and selectivity. availability and expression of toxic activity. Or, in some Boththesensitivityandselectivityofspectroscopicdetec- cases,surfacecoatingmaterials mayhaveaprophylactic torsaredependent, inpart,onwavelength(s)employed. effect on expression of the toxicity of the underlying In addition, it is desirable to choose a detector with bulk of the particle. An example of the latter effect a wide linear working range to be able to assess both is the modified activity of the quartz component of majorandminorchemicalcomponentsintheworkplace some mixed composition mineral dust exposures for environment.Inmonitoringtheworkplaceenvironment, causing mixed dust pneumoconioses. SEM with EDX the most commonly employed HPLC detectors are the orwavelengthdispersiveX-rayspectroscopycanprovide UV/VISandfluorescentphotometers,whereas,themost someinformation abouttheelementalcomposition with commonlyusedGCdetectorsaretheflamephotometric depth into a particle, by acquiring X-ray spectra at detector(FPD)andthemassspectraldetector(MSD). two or more electron beam accelerating voltages. This With the increased use of highly toxic gas and method has been used to detect thin submicrometer vapor mixtures in science and industry, FTIR and aluminosilicate coatings or clay ‘‘surface occlusion’’ on Raman spectroscopies have played an important role respirable quartz particles. Applications of SEM to in industrial hygiene monitoring. Although FTIR and the study of pneumoconioses, including determination Ramanspectroscopiesaresimilarinthatbothtechniques of lung particulate burden, particle chemistry, and provide information on vibrational frequencies, there respirableparticlesurfacecharacterization,aredescribed are many differences between the two techniques. insection3ofthisarticle. For example, some vibrations are only Raman active Atomic spectrometric methods continue to be used while others are only IR active; the vibration of a widely for occupational health evaluation of inorganic heteropolar diatomic molecule is IR active, whereas metals and are documented briefly in section4. The that of a homopolar diatomic molecule is not IR developmentofICPAEStechniqueshasbeenincreasing, active. The method for determining quartz content in and has been applied to elemental analysis of nearly all respirable coal dust is often based on dispersive IR the elements. ICPAES offers a simultaneous or rapid or FTIR spectroscopy. Like IR, RS is a powerful sequential multielement determination capability at the technique and has a variety of applications. They are major, minor, and certain trace concentration levels. It complementary,andbothareutilizedwheneverpossible. has become established as a widely accepted methods IRandRamanspectroscopesandtheirrelatedtechniques for the analysis of metallic aerosols, powders of metals, andapplicationsarediscussedbrieflyinsection6. dusts, and fly ashes. A vast amount of published atomic Finally, field-portable methods, which have received spectrometric methodology is available, covering a wide increasing attention, are summarized in section7. Field- rangeofapplicationareas.Thisisbecausetraceelements portable methods for monitoring airborne workplace released into the atmosphere in industrial processes contaminants and toxins have received increasing atten- have aroused great interest. Major advantages include tion. A number of portable monitors for airborne specificity,speed,andeaseofuse.However,forultratrace contaminantshavebeencommerciallyavailableformany or trace level analysis atomic spectrometric methods years,butnewdevelopmentsmayprovideforon-sitecom- have been increasingly replaced by inductively coupled pliancemonitoring,whichhasheretoforebeenmorethe plasma mass spectrometry (ICPMS). AS and its related exceptionthantherule.Theabilitytoconductmeasure- techniquesandapplicationsaredescribedinsection4. mentson-siteintheoccupationalsettingofferssignificant Spectrophotometric detectors coupled to chromato- advantages.Field-portable methods areoften desiredso graphicseparationtechniquesareoftenusedtocharacter- that decisions regarding worker protection, engineering izetheworkplaceenvironment and arediscussedbriefly controls, and so on can be made quickly. The capability in section5. These techniques are employed to confirm for rapid decision-making offered by on-site monitor- theaccuracyofdirectreadinginstrumentsorwhendirect ing can help to save costs, and also offers a means to reading instruments are not available for the environ- assess, and thereby prevent, worker overexposures to mental contaminant, when the workplace environment toxicsubstancesinatimelymanner.Field-basedmonitor- is complex and contains multiple chemicals that need to ingisespeciallyusefulforapplicationsintheconstruction be measured, for regulatory documentation of exposure industry, in agriculture, and in other situations where levels, and for biological monitoring. Spectrophoto- jobs may be short-term and the workforce is transient. metric detectors that are coupled to high-performance On-site techniques can also be beneficial in instances 4 INDUSTRIALHYGIENE whereshort-termmonitoringisdesired.Insection7,field- addition, the coupling of MSwith separation techniques portablespectrometrictechniquesarecovered,andsome and the development of new ionization methods such applicationsarepresented. aselectrosprayionization,APCI,andMALDIhavecon- To date, highly specific, selective, and sensitive spec- tributedtogreatstridesforwardinthisfield.Theabilityto troscopictechnologieshaveallowedforthedevelopment ionizepolar,labileandinvolatilespecieshasbeenthefun- ofnovelmethodologiesandnewindicatorsofexposures. damental basis upon which MS extends its applications. Therefore, the assessment of the actual body burden of It is important for us to recognize the breakthroughs chemicals, which is directly related to potential adverse in ionization techniques of the 1980s and 1990s. These occupationalhealtheffects,canbeaccomplished. advanceshaveallowedapplicationofMStoprogressand developrapidly. The interest in using MS by scientists in studies per- tinent to industrial hygiene is driven by the need to 2 MASSSPECTROMETRY understand the basic physical, chemical, and biologi- cal processes related to workers’ exposure to hazards 2.1 Introduction and to devise new methodologies to control exposures The mass spectrometer is an instrument capable of in the work environment. MS is capable of looking at producingabeamofionsbyconvertingneutralmolecules the details of exposure, and is a highly sensitive tool into gaseous ions, and then separating these ions for structural determination of aberrant proteins, devel- accordingtotheirmass-to-chargeratioandrecordingthe opment of biomarkers for exposures, identification of relativeabundancesoftheseparatedionspeciesasamass microbialproductsincomplexsamples,andcharacteriza- spectrum..1/TodayMShasbroughtspecialcapabilitiesto tionofhazardsinoccupationalandenvironmentalhealth awidevarietyofscientificresearchbyprovidingspecific evaluations. analyses of substances, their metabolites, and biological macromolecules, often with structural information. The 2.2 GasChromatography/MassSpectrometry rangeofapplicationsincludesqualitativeandquantitative 2.2.1 PrinciplesandInstrumentation analyticalproceduresemployedinenvironmentalhealth andindustrialhygiene. ThecouplingofGCwithMSwasfirstachievedin1957..5/ Ininstrumentation, the massdetector has been devel- It is a combination of two microanalytical techniques: oped in a variety of types, shapes, and sizes..2–4/ The a separation technique, GC, and an identification tech- selectionofthedetectorisbasedontheneedsoftheuser nique, MS. The gas chromatography/mass spectrometry anditsfunctions:.1/ thequadrupoleanalyzerisemployed (GC/MS)combinationovercomescertaindeficienciesor toprovideanelectronionization(EI)orchemicalioniza- limitations caused by using each technique individually, tion (CI);.2/ the ion trap spectrometer, which is a highly and gives a two-dimensional identification consisting of geometricallymodifiedquadrupoleanalyzer,isadequate both a GC retention time and a mass spectrum for each for low-energy collision;.3/ the ion cyclotron resonance component of the mixture. This combination has sev- spectrometer, which is the basis for Fourier transform eral advantages. First, it can separate components of a MS,hashighresolution;.4/ thetime-of-flight(TOF)spec- complexmixturesothatmassspectraofindividualcom- trometer,whichseparatesionsintimeratherthanspace poundscanbeobtainedforqualitativepurposes;second, and has an almost unlimited mass range, is used for it can provide quantitative information on these same detection of macromolecules; and.5/ the magnetic sector compounds. GC/MS can provide a complete mass spec- instrument, in which ion separation is achieved spatially trum from as little as 1pmol of an analyte, which gives bytheapplicationofamagneticfield(sometimescoupled directevidenceforthemolecularweightandacharacter- withanelectrostaticfield),issuitedtoconductcollision- istic fragmentation pattern or chemical fingerprint that ally activated dissociation at high energy. Furthermore, can be used as the basis for identification. Although the bycouplingoftwoormoretypesoftheabove-mentioned direct GC/MS method is limited to the analysis of those mass analyzers, an MS/MS or an ion trap mass spec- compounds that can be made volatile without thermal trometry (MSn) technique is achieved. MS/MS is a key decomposition, many compounds that are nonvolatile instrumentaldevelopmentinanalyticalandbioanalytical can be handled successfully after chemical derivatiza- chemistry. It is widely applied in the characterization of tion. The instrumentation of GC/MS consists essentially molecularstructuresandinthetraceanalysisoftargeted of three components: the gas chromatograph, the mass analytes.Themostwidelyusedactivationmethodiscalled spectrometer and a data system. GC/MS has developed collisionallyinduceddissociation,inwhichprecursorions intooneofthemostsensitiveandselectiveanalyticaltech- areselectedinthefirstMSofmultistageMSforrepulsive niquesfortheseparation,identificationandquantification collisions with inert gases such as helium or argon. In ofcomponentsofcomplexmixtures. 5 SPECTROSCOPICTECHNIQUESININDUSTRIALHYGIENE The GC/MS technique has been utilized in a diverse monomericsugarsandaminoacids.Samplepretreatment range of applications such as toxicology, environmental ofmicroorganismsforGC/MSanalysisgenerallyinvolves monitoring,molecularbiology,clinicalhealth,andindus- extractionoftheclassofcompoundsofinterest(e.g.lipids, trialhygiene,aswellasmanyothers.Intheseapplications, proteins,orcarbohydrates)followedbyderivatization. the analyte is often present in a complex matrix consist- ingofagreatnumberofcompoundswhichmaymaskits 2.2.2.2 Electron Ionization and Chemical Ionization presence or otherwise inhibit its detection. One of the EI is one of the main ionization methods employed mostcommonwaysinwhichGC/MSisusedinindustrial in GC/MS system,.8/ while CI has also been employed hygieneisasadiagnostictool,whichisparticularlyuseful occasionally. In EI, energy sufficient for ionization and in the analysis of complex mixtures where the analytes fragmentation of the analyte molecules is acquired by arepresentinlowquantities.GC/MSisalsowidelyused interactionwithelectrons(ca.70eV)fromahotfilament. indetermininghazardousmaterials,derivedmetabolites, Some structural features of the analyte molecule can be andprotein/DNAadductsinbiologicalfluids(e.g.blood deduced from the fragmentation pattern of the molecu- orurine)fromworkersexposedtospecifichazards. lar ion. During the ionization process, in addition to the productionofpositiveions,asmallnumberofmolecules undergoadditionofoneormoreelectronstoformnega- 2.2.2 Methodology tiveions.Attheoperatingionizingenergyoftheanalytical 2.2.2.1 Sample Preparation Sample preparation is massspectrometer(60–100eV)thesensitivityofnegative important for successful analyses by GC/MS. Industrial ionformationisseveralordersofmagnitudelessthanthat airborne hazards include gases, vapors, liquids, and par- forpositiveionproduction.ComparedwithEI,CIisasoft ticulates. Air sampling is of course a crucial step within ionizationtechnique.Itachievesionizationoftheanalyte the total scheme of air analysis. There are a variety of by collision with reagent ions (usually proton-rich ions), methodsavailableforthecollectionofairborneparticles. but without transferring excessive energy to the nascent Generally, a method selected will often depend on the analyte ions. The result is the formation of abundant purpose for which the sample is being taken, and also adduct ions, often protonated molecules, that contain dependonthetypeofcompoundstobeanalyzed.Afew the intact molecular species of the analyte. In this way, preprocessing steps may be needed to manipulate the CI and EI are complementary. Because of the soft ion- sample into a form ready for analysis. In addition, the izationprocess,theeven-electron molecular adduct ions physical state of sample material will affect the method undergo little fragmentation compared with that of the to be used for the introduction of sample into the spec- odd-electron molecular ion produced during EI. Often, trometer. A wide variety of techniques are available for CI spectra of individual analytes are sufficiently simple processing gaseous, liquid and solid samples. In indus- to allow the direct analysis of mixtures, and therefore trial hygiene studies, one prevalent strategy in dealing canbeusedtoprovidestructuralinformation thatisnot with air samples consists of filter collection of particles, availablefromanEIspectrum. followed by an appropriate second stage adsorption of organic chemical vapor or fumes onto XAD-2 sorbent 2.2.2.3 QuantitativeAnalysisandSelectedIonMonitoring [treated with 2-(hydroxymethyl)piperidine] or charcoal ThequantitativeapplicationsofMSarebasedoncompar- tubes, and then solvent desorption and GC/MS analy- isonbetweentheioncurrentobtainedfromtheanalytein sis. There have been considerable improvements in air thesamplematrixandtheioncurrentfromanothercom- samplingandGC/MSdetection..6;7/ poundchosenasaninternalstandard,ortheioncurrent Another commonly used strategy of GC/MS analysis obtained from analyses of standard aliquots of the pure is the derivative method. Derivatization is an approach analyte.Themethodofrecordingtheioncurrentisusually forincreasingvolatilityoftargetanalyteswhichispartic- byselectedionmonitoring(SIM),althoughthetechnique ularlyusefulforbiologicalsamples.Inthosecaseswhere ofrepetitivescanningoveranarrowmassrangeissome- compounds are too polar or thermally unstable to be timesemployed.ThetechniqueofSIMisoneofthemost amenable toGC analysis,thesituation canbe improved versatile and commonly used spectrometric methods. It by the formation of a suitable derivative with character- allowsamassspectrometertorecordsimultaneouslythe istics that render it more amenable to GC. In addition, intensities ofalimited number ofchosenions only.This derivatization can enhance sensitivity and selectivity by allows the instrument to dwell for a greater proportion altering the fragmentation mechanism of the molecule. of the analysis time on those mass-to-charge ratios of Afurtherbeneficialeffectofderivatizationisthatitnor- greatestsignificanceinthemassspectrumoftheanalyte mallyresultsinacompoundpossessingcharacteristicions ofinterest,resultinginanincreaseinsensitivity.Amajor atmass-to-chargeratios.Intissueanalysis,thesampleusu- application of this technique is to provide quantitative allyrequiresdialysisafterhomogenizationtoremovefree analysisofcompoundsatlowconcentrations..9/ 6 INDUSTRIALHYGIENE 2.2.3 Applications PAHswereanalyzedbyGC/MSingraphitedmineraloils used for mold lubrication and in aerosols emitted dur- In the 1990s, the application of GC/MS and related ing their application in two plants. High boiling PAHs techniques for the characterization and quantitation of were detected in oils but generally not in air. Volatile organiccompoundsandbiomarkershasgrownspectacu- PAHs were found in oil samples and to a lesser extent larlyinenvironmentalhealthandoccupationalexposure in air near the emission source. Additionally, Bundt studies. One such example is the use of GC/MS in the analysis of the health effects of the environmental aro- etal..13/ have investigated structure-type separation of matic hydrocarbons. Polycyclic aromatic hydrocarbons dieselfuelsbysolid-phaseextraction(SPE)andidentifi- (PAHs)areubiquitous,andsomeofthemarepotentially cationofthetwo-andthree-ringaromatics.Commercially carcinogenicsubstances,towhichhumansareexposedin available standards were used for identification. Sulfur- theenvironmentandincertainworkplaces.Estimationof containing PAHs in diesel fuel are mainly represented theresultinghealthriskisthereforeofgreatoccupational bymethyl-substituteddibenzothiophenes.Cooper.14/has healthimportance.ThedeterminationofPAHsandtheir developedaGC/MSmethodtoconfirmthepresenceofN- metabolitesorprotein/DNAadductsasbiomarkersisthe nitrosaminesinworkplaceairsamples.Detectionlimitsof mostsuitablewaytoassesstheexposureandtoestimate thethreeproceduresandretentiontimeprecisionofboth thisrisk. SIMtechniquesaregood.Typicalexamplesoftheuseof Sturaroetal..10/ havereportedaGC/MSinvestigation thesetechniques for confirmation of N-nitrosamines are of polycyclic aromatic compounds (PACs) in the man- described by Cooper..14/ More applications are summa- ufacture of rubber tubes, using a two-stage air sampler rizedinTable1. and aGC/MS method to monitor eight PACs. With this Urinary naphthols (1- and 2-naphthol) have been method, detection limits of 8–15ngmL(cid:0)1, correspond- suggested as route-specific biomarkers for exposure to ing to a level of 12–23ngm(cid:0)3 in the workplace, were airborne PAHs..27/ The application of urinary naphthol achieved.Workersexposedtoairbornearomaticamines levels as biomarkers in 119 Japanese male workers inanotherrubbermanufacturingprocesswereevaluated was reported. The urinary 1- and 2-naphthol levels by Menichini etal..11/ Samples were collected on a glass were observed three and sevenfold higher, respectively, fiber filter, processed through a silica gel tube, and ana- amongsmokersthanamongnonsmokers.Alsotheratios lyzed by GC/MS/SIM. Their work outlines a procedure of urinary 2-naphthol to 1-naphthol were significantly for the determination of occupational exposure to air- higher among smokers than nonsmokers. In another borne aromatic amines in the rubber industry and the study, a method for the simultaneous determination of applicationofthismethodinatiremanufacturingplant. urinary phenanthrene, fluoranthene, pyrene, chrysene In related work, Menichini etal..12/ determined PAHs and benzo[a]pyrene metabolites has been developed in mineral oils and oil aerosols in glass manufacturing. for individual risk assessment at a PAH-burdened Table1 TypicalapplicationsbasedonGC/MSmethods No. Chemicalhazards Sampletype Ref. 1 PAHs(glassmanufacturing) Mineraloilsandoilaerosols 12 2 PAHsindieselfuel Petroleum 13 3 PAHs(cookingplantandfoundries) Workplaceair,anddust 21 4 PACs(energylaboratory) Coalcombustionproduct 16 5 PACs(manufactureofrubbertubes) Workplaceair 10 6 Perchloroethylene(fivedrycleaningfirms) Workplaceair,andbloodofexposedworkers 19 7 Airbornearomaticamines(rubber Workplaceair 11 manufacturing) 8 N-Nitrosamines Workplaceair 14 9 VOCs Indoorair 18 10 Polychlorinatedbiphenyls(electricalworkers) Workroomsurfaces,tools,palms,andblood 22 11 Polybrominateddioxins,dibenzofurans, Combustion,andflameretardants 20 octachlorostyrene 12 Glycolethers(newspaperprintingplant) Workplaceair 17 13 Organicvaporpollutants Ambientatmospheres 15 14 Resinacidcompounds Emittedfromrosininsolderingflux 23 15 Semivolatileorganiccompounds Workplaceatmospheres 24 16 Airbornechemicalagents Air 25 17 Coaltarpitchvolatiles Coldtarpitchfume 26 VOCs,volatileorganiccompounds. 7 SPECTROSCOPICTECHNIQUESININDUSTRIALHYGIENE workplace..28/ The method allows the determination of and CI with isobutane as the reagent gas. The use of 25 different components. The PAH exposure of coke Hb and serum–protein adducts of hazard reagents as plantworkersduringseveralconsecutivedaysresultedin biomarkers for occupational and environmental expo- fairly constant individual urinary metabolite profiles..29/ sure assessment has received increasing interest. The It was also demonstrated that in the case of coke plant environmental pollutant 2,4,6-trinitrotoluene (TNT) is workersthereisacorrelationbetweeninhaledPAHsand animportantoccupationalhealthhazard,andistakenup metabolitesexcreted.Massrelationshipsbetweeninhaled throughtheskinandbyinhalation.Itisthereforeessen- PAHsandmetabolitesexcretedwerefoundtodifferfrom tialto havefastand reliablemethods to monitor human oneindividualtoanother. exposure. In a related work, a GC/MS method, which The exposures of agricultural workers to organochlo- quantifies Hb adducts of TNT for 50 workers and con- rine pesticides were studied by Guardino etal..30/ The trols from a Chinese munition factory, was reported by chlorinated pesticides and their metabolites in whole Sabbioni etal..36/ The Hb adduct levels ranged from 3.7 blood samples from 30 farmers and 24 nonoccupation- to522ng.However,incontrolsamplesnoadductscould ally exposed workers were determined by GC/MS. The be found. In another study,.37/ alachlor–protein adducts potential that sawmill workers might be exposed to wereexaminedaspotentialbiomarkersofalachlorexpo- chlorophenols was investigated by Kontsas etal..31/ A sure,agenotoxicandcarcinogenicherbicide.Themethod GC/MS procedure for the determination of chlorophe- developedwasbasedontheobservationthatcleavageof nols in urine was developed. The concentrations of S-cysteinylalachlor–proteinadductsbymethanesulfonic urinary chlorophenols in previously exposed workers acidgavetherearrangementproduct. were of the same magnitude as those found in non- Hbsamplesfromethyleneoxide-exposedworkersand exposed controls and in the general population. The nonexposed referents were analyzed by Farmer etal..38/ feasibility of using plasma, blood and hemoglobin GC/MSwasusedtodetermineanHbadductasitsmethyl (Hb) adducts for monitoring occupational exposure to ester heptafluorobutyryl derivative, after hydrolysis of the suspected human carcinogen 4,40-methylenebis(2- the protein and isolation of the alkylated amino acid. chloroaniline) (MOCA) was investigated by Vaughan Ranasinghe etal..39/ have reported an application of etal..32/ The levels of MOCA in the blood and urine of GC/electroncapturenegativeCIhigh-resolution MSfor fiveindividuals whowereexposedtoMOCAduringthe characterization and quantitation of DNA and protein manufactureweredeterminedbytheGC/MSmethod.It adducts.Themethodhasadequate sensitivityandspeci- was found that the use of blood samples for monitoring ficitytomeasureaccuratelyDNAandproteinadductsas exposuretoMOCAoffersadvantagesoverthecurrently low as endogenous concentrations in rodent and human usedurinaryMOCAmeasurements. tissues.AdditionalapplicationsarelistedinTable2. Occupational exposure to toluene diisocyanate (TDI) among workers in a polyurethane foam factory.33/ was 2.3 LiquidChromatography/MassSpectrometry,Liquid studied during48-hperiods and biological samplesfrom ChromatographyTandemMassSpectrometryand nine subjects. Five workers were found to show high LiquidChromatographyIonTrapMass average urinary elimination rates of TDI. The elimina- Spectrometry tionratecurvesforallofthesubjectsstudiedhadalinear relationship with exposure to TDI. The study indicates 2.3.1 PrinciplesandInstrumentation that it is possible to monitor exposure to TDI by moni- toringurinaryconcentrationsofTDIbyGC/MS.Astudy Thehistoryofliquidchromatography/massspectrometry of exposure to benzidrine was reported by Hsu etal..34/ (LC/MS) starts in the early 1970s,.62/ and since then Exposuretobenzidine,whichissubsequentlyacetylated the technique has been developed rapidly. There are to N-acetylbenzidine and N,N0-diacetylbenzidine, has severalgeneralreviews.63–66/andbookspublishedonthis beenimplicatedinthedevelopmentofbladdercancerin subject..67–70/ The combination of LC and MS offers humans.Inthisstudy,anisotopedilutionGC/negativeion the possibility of taking advantage of both LC as a CIMSmethodwasdevelopedtoquantitateurineconcen- powerful separation technique and MS as a selective trations ofbenzidine andits acetylatedmetabolites. The andsensitivedetector.AconsiderablenumberofLC/MS method is applicable to the measurement of other aro- interfaces have been developed. Widely used interfaces matic amines and their acetylated metabolites. Worker included particle beam, thermospray, continuous-flow exposure to sawing fumes from pine was investigated fastatombombardment(FAB),electrosprayandAPCI. by Eriksson etal..35/ Three metabolites from a-pinene Electrospray ionization was one of the most important have been identified in human urine after occupational ionization methods in the 1990s, and is extensively used exposure to it. Urine was enzymatically hydrolyzed, inLC/MSsystems.Electrosprayistheresultofcharging and metabolites were identified by GC/MS using EI aliquidataneedletipbyapplyingahighpotential.With 8 INDUSTRIALHYGIENE Table2 TypicalbiomonitoringbasedonGC/MSmethods No. Biomarkers Sampletype Ref. 1 Alachlor–proteinadducts(alachlorexposure) Invitroandinvivo 37 2 Hbadducts(ethyleneoxideexposure) Blood 38 3 Hbadducts(methylbromideexposure) Invitroerythrocytes 40 4 Trinitrotolueneandmetabolites Urine 42 5 Hbadducts(3,3-dichlorobenzidineexposure) Raterythrocytes 43 6 PAHsandmetabolites(PAH-exposedworkers) Urine 28 7 DNAadducts(2,3-epoxy-4-hydroxynonanalexposure) CalfthymusDNA 44 8 Metabolites(monoterpenesa-pineneandb-pineneexposure) Urine 35 9 Hbadducts(TNT) Blood 36 10 Benzidineandmetabolites(benzidineexposure) Urine 34 11 Hb[(4,4-methylenebis(2-chloroaniline)] Blood,plasma,andurine 32 12 Hbadductsandmetabolites(MDIexposure) Urine 45 13 PAHs(atvariousworkplaces) Urine 29 14 DDTandrelatedcompounds(agriculturalworkers) Wholeblood 30 15 Chlorophenols(sawmillworkers) Urine 31 16 VOCs Wholeblood 41 17 MDAandmetabolites(MDAexposure) Urineandbloodplasma 46 18 Hbadduct Raterythrocytes 47 19 Pesticidemetabolites(pesticidesexposure) Urine 48 20 N-Phenylvaline(benzeneexposure) Blood 49 21 S-Benzyl-N-acetyl-L-cysteine(tolueneexposure) Urine 50 22 DNAdamage(benzo[a]pyreneexposure) Humanlymphocytes 51 23 Toluenediamine(TDI) Urineandplasma 33 24 Polyaromaticcarcinogen–DNAadducts Review(humans) 52 25 Hbadducts(PAHexposure) Blood 53 26 Phenolandmetabolites(phenolexposure) Urineandplasma 54 27 Hbadducts(ethyleneoxideexposure) Blood 55 28 S-(2-Carboxyethyl)cysteine(acrylamideexposure) Blood 56 29 DNA–proteinadducts(PAHexposure) Review 57 30 DNAadducts(traceorganicexposure) Review 58 31 PAH–DNAadducts(PAHexposure) Review 59 32 DNAadducts(exposureassessment) Review 60 33 DNAadducts(chemicalcarcinogenesis) Review 61 DDT,1,1,1-trichloro-2,2-bis[p-chlorophenyl]ethane;MDI,methylenediphenyldiisocyanate;MDA,methylenebisaniline. the increase in the potential, the droplet size is reduced offers a major advantage for structural characterization andthedropletsbegintohaveahorizontalcomponentin of components in mixtures and trace analysis. Success- theirmovementaswellasahigherspeed.Nowadaysmost ful interfacing of this technique has created an effective typesofatmosphericpressureionsourcescanbeusedin methodology in the analysis of nonvolatile, labile, and combinationofanelectrosprayandAPCIinterface,which macromolecularcompounds.Awidevarietyofimportant isthemostwidelyadaptedinterfacetoLC/MSsystems. applications include determination of large portions of Asahyphenatedtechnique,LCcouplingtoMSresults proteinsequenceswitheitheron-lineoroff-lineenzymatic inapowerfuland versatileanalytical tool. Furthermore, hydrolysis, and development of molecular biomarkers, LC coupling to a tandem or an ion trap mass spectrom- which represent a more accurate determinant of poten- eter (LC/MS/MS or LC/MSn) plays another important tialriskofexposuretohazardsorcarcinogensthanthose role in the development of new analytical strategies. whichonlyassessexternalexposure. MS/MS, generally, includes a collision cell in which deliberatefragmentationofparentionscanbeachieved. 2.3.2 Methodology Collision-induced fragmentation provides daughter ions by collisional energy transfer between parent ions and a 2.3.2.1 SamplePreparation Bothon-lineandoff-line collision gas, normally helium, nitrogen or argon, at an samplepretreatmentbySPEarecommonlyemployedin elevated pressure.The mostwidely used MS/MS config- LC/MS, LC/MS/MS, and LC/MSn methods. In quantita- uration is the triple quadrupole instrument, where mass tive studies of metabolites, and protein–DNA adducts, analysis is performed in the first and third quadrupole off-linesamplepretreatmentappearstobepreferred.This whilethesecondquadrupoleisusedasacollisioncell.It is partly due to the composition of biological samples, 9 SPECTROSCOPICTECHNIQUESININDUSTRIALHYGIENE where the presence of proteins may cause clogging of example, direct analysis of DNA adducts by using LC theSPEcolumnsorcartridgesused.Forexample,atyp- combined with electrospray ionization MS can obviate ical Hb and DNA adducts assay involves extraction of thoseproblemsarisingfromtheemploymentofchemical DNA or Hb from its biological matrix (e.g. blood or derivatizationneededforGC/MS.ThatisbecauseDNA tissue), cleavage of the macromolecule to smaller com- adducts are usually very polar, and the derivatization is ponents, and the use of enrichment techniques, such as often difficult and less successful. A number of studies derivatization and/or SPE preconcentration. To speed havedemonstratedthatprotein–DNAadductsinbiolog- up a complete procedure, the sample pretreatment can ical samples can be quantified precisely and accurately be performed on an automated batch-scale SPE. Some- by using LC coupling to MS or MS/MS..71–75/ Workers times, improving the speed and/or performance of the are occupationally exposed to a wide array of chemi- samplepretreatmentpriortoLC/MSanalysisisamatter calcompounds.Thecompoundsortheirmetabolitescan ofconsiderableresearch. interactwithbiologicalmacromoleculessuchasproteins, After determination of the molecular mass and the RNA, and DNA. These interactions can result in cova- elemental composition, the next step in the qualitative lentbondingbetweenthechemicalsandmacromolecules, analysis is the interpretation of fragment ion peaks in leading to DNA damage and the formation of DNA the mass spectrum to achieve structure elucidation. A adducts.Ifthesedamagesarenotenzymaticallyrepaired, powerfultoolinstructureelucidationistheuseofMS/MS. they can be the cause of mutations and might lead to The information from a measured spectrum is reduced chemically induced carcinogenesis. The structural eluci- toasmallnumberofthemostsignificantpeaksandthen dation of these DNA adducts is an important research comparedwiththelibraryspectra.Computerizedlibrary topic in cancer prevention. The use of DNA adducts searching is very useful as it provides ideas on which as biological markers for risk assessment and occupa- direction to search when a completely unknown analyte tionalandenvironmentalmonitoringhasgeneratedgreat must be identified, or provides adequate confirmation interest because they represent direct indications of pri- whenthepresenceofacompoundistobeconfirmed. marydamagetogeneticmaterialbychemicals.Therefore, protein–DNA adducts may prove to be more accurate 2.3.2.2 InterfaceTechnology Inthe1970s,1980sand andreliablethanmeasurementofexternalexposure.To 1990s,LC/MSdevelopmenthasresultedinaconsiderable accomplish these goals, a sensitive and specific LC/MS number of different interfaces. A major effort for methodiscapableofdetectinglowpicomolequantitiesof this research was to improve the instrument capability adductinrelativelysmallcomplexbiologicalsamples,and to ionize analytes including highly polar, labile and isspecificenoughtoconfirmthestructureoftheadducts. biomacromolecules directly from the liquid phase. The The coupling of LC to MS and MS/MS pertinent to most widely used interfaces are electrospray, APCI, industrialhygienestudieshasbeenreportedbyanumber thermospray, particle beam, and continuous-flow FAB. ofresearchers.SomeofthemaresummarizedinTable3. Detailedinterfacingstrategiesareavailablefromabook In one study,.76/ sensitive and specific isotope dilution by Niessen..62/ The electrospray interface provides one LC/MSandLC/MS/MSmethodsweredevelopedforthe of the most promising interfaces for LC/MS in that it detectionandquantitationofDNAadductsformedupon disposesofthemobilephaseduringspraygenerationand exposure of animals to carcinogenic 1,2-dihaloethanes, offersdetectionlimitsintothefemtomolerange.Ithasthe 1,2-dichloroethane and 1,2-dibromoethane. These are followingadvantages:(1)directionizationfromsolution; important industrial chemicals used as additives in (2)productionofmultiplychargedionswhichextendthe gasoline, as intermediates in the production of vinyl effectivemassrangeofthemassanalyzer;(3)introduction chloride,vinylbromide,andotherhalogenatedorganics, ofmethodstoaidindesolvationoftheanalyte;and(4)low ascomponentsofgrainorsoilfumigants,andassolvents backgroundfromtheionizationprocess.Itisremarkable for cleaners and other industrial products. In another thatwecanobtainthemassspectrumofaproteinwitha study,anHPLC/electrosprayMSmethodwasdeveloped largemolecularmass,andconsumeanamountofsample for the analysis of 7-(2-hydroxyethyl)guanine, the major of the order of picomoles to femtomoles. APCI is also DNA adduct formed after exposure to ethylene oxide. usedforcontinuousmonitoringofanHPLCcolumn.The ThemethodisbasedonDNAneutralthermalhydrolysis, vaporized eluate from the HPLC is forced through the adduct microconcentration, and final characterization APCIsourcewithaslowheatedstreamofnitrogengas. and quantification by HPLC coupled to single-ion monitoringelectrosprayMS.Themethodwasfoundtobe 2.3.3 Applications selective, sensitive, and easy to handle with no need for Application of LC/MS in the analysis of workplace enzymatic digestion or previous sample derivatization. and environmental samples has been increasing, espe- Yen etal..77/ have developed a method to quantify N,3- cially in the development of exposure indicators. For ethenoguanine, a promutagenic DNA adduct of vinyl 10 INDUSTRIALHYGIENE Table3 TypicalapplicationsbasedonLC/MS,LC/MS/MS,andLC/MSnmethods No. Chemicalhazardsand/orbiomarkers Methods Ref. 1 DNAadduct(1,2-dichloroethaneand1,2-dibromoethaneexposure) LC/MS/MS 76 2 Benzenemetabolites(benzeneexposure) LC/MS/MS 82 3 Aromaticsulfonates(textileindustryorconstruction) LC/MS 71 4 CPandIF(healthcarepersonneloccupationallyexposure) LC/MS/MS 81 5 DNAadducts(1,3-butadieneexposure) LC/MS/MS 80 6 Hbadducts(methylbromideexposure) LC/MS/MS 83 7 DNAadducts(treatedwithN-nitrosodiethylamine) LC/MS 79 8 PAH–DNAadducts(invitroreactionwithPAHs) LC/MS 72 9 DNAadducts(invitroreactionwithbisphenolAdiglycidylether) LC/MS 78 10 Ethyleneoxide–DNAadduct(ethyleneoxideexposure) LC/MS 73 11 DNAadducts(vinylchlorideexposure) LC/MS 77 12 PAHmetabolites(PAHexposure) Review 74 13 DNAadducts(invitroreactionwithbisphenolAdiglycidylether) LC/MS/MS 75 14 Hazardousindustrialchemicals LC/MS 85 15 Aliphaticisocyanates LC/MS 86 16 PolymericMDIandotherisocyanates LC/MS 87 17 TDI LC/MS 88 CP,cyclophosphamide;IF,ifosfamide;MDI,methylenediphenyldiisocyanate. chloride exposure. The applicability of the method was Benzene is an important industrial chemical and established by determining DNA adduct in rats treated ubiquitous environmental pollutant. It is used in the withchloroethyleneoxideandanunexposedhumanliver. manufacturing of a wide variety of consumer products. It was observed that the concentration of DNA adduct Melikian etal..82/ developed a sensitive and specific in the rat livers increased with increasing dose, but was LC/MS/MS assay for determination of urinary benzene inversely related to the time after exposure. This trend metabolites.Theobjectiveofthisstudywastoinvestigate suggestsrapidDNArepairandthatadductsreduceinrat how various levels of exposure affect the metabolic livers. activationpathwaysofbenzeneinhumansandtoexamine Vanhoutteetal..78/ developedananoscaleLC/electro- the relationship between urinary metabolites and other spray MS methodology for the detection and identifi- biologicalmarkers.Ferrantietal..83/usedLC/MS/MSfor cation of DNA adducts by in vitro reaction mixture the structural study of adducts formed in human Hb resulting from the interaction of calf thymus DNA with by in vitro exposure of erythrocytes to the alkylating bisphenolAdiglycidylether.Inotherwork,Singhetal..79/ agent methyl bromide (MeBr). MeBr is a highly toxic developed an HPLC/electrospray MS method to detect gas widely used as a fumigant of field soil for control and characterize two major ethylated DNA adducts for of a wide spectrum of pests and diseases. Peptide monitoringexposuretogenotoxicethylatingagents.This mapping by this method allowed location of methylated approachwasshowntobecapableofdetectingtheDNA amino acids within the protein sequence. The results adductinlivertissuefrommicetreatedintraperitoneally demonstrated the usefulness of the analytical approach with N-nitrosodiethylamine. A major chemical in rub- for the characterization of Hb adducts with methyl ber and plastics manufacture, 1,3-butadiene, inducing bromideorsimilarcompounds, whichcanconstitute the DNA adducts in vivo and in vitro, was investigated by Tretyakova etal..80/ The LC/electrospray MS/MS meth- basisforbiomonitoringofhumanexposure. The LC/MS, LC/MS/MS, or LC/MSn techniques have ods developed in this work provide the means to study been developed for characterization and quantitation of accumulation,repairanddose–responserelationshipsof pesticides, herbicides, and insecticides..84/ For example, 1,3-butadiene–DNAadductsinvivo. Occupationalexposuretocyclophosphamide(CP)and carbamate pesticides are used in large quantities. Since ifosfamide (IF) was investigated by Minoia etal..81/ An theirthermalliabilityprohibitsGCanalysis,theanalysis LC/MS/MS system was employed to monitor CP and of these compounds and their metabolites is usually IF exposure of 24 workers. The extent of exposure was performed by LC/MS. They have been analyzed using assessed by the analysis of air samples, wipe samples, most commercially available interfaces. Positive ion dermalpads,andurinaryexcretionatthebeginningandat detection with a soft ionization technique is the method theendoftheworkshift.Theresultsofthisinvestigation of choice. Phenylurea herbicide is one of a group of demonstratethathigherriskmaybecausedbyincorrectly herbicides which are frequently analyzed by LC/MS or usingairflowhoods. LC/MS/MS..84/Chlorinatedphenoxyacidherbicideshave

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