Spectroscopic Techniques in Industrial Hygiene

42 Pages·2000·0.289 MB·English

by  Wang J., Siegel P.D., Lewis D.M.

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