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Review!

Modern Instrumental Methods in Forensic Toxicology*Michael L. Smithl,t, Shawn P. Vorce1, Justin M. Hollerl, Eric Shimomura1, Joe Magluilo1,Aaron). Jacobs1,2, and Marilyn A. Huestis31Division of Forensic Toxicolog)'t Office of he ArmedForces Medical Examiner, ArmedForces Instituteof Patholog)'t1413 Research Blvd., Bldg. 102, Rockville, Maryland 20850; 2Army Medical Department Board, Fort Sam Houston,Texas 78234; and 3Chemistry and Drug Metabolism, IntramuralResearch Program, National institute on Drug Abuse,National Institutes of Health, 5500 Nathan Shock Drive, Baltimore, Maryland 21224

IAbstract IThis article reviews modern analytical instrumentation in forensictoxicology for identification and quantification of drugs and toxinsin biological fluids and tissues. Abrief description of the theoryand inherent strengths and limitations of each methodology isincluded. The focus is on new technologies that address currentanalytical limitations. A goal of this review is to encourageinnovations to improve our technological capabilities and toencourage use of these ana lytical techniques in forensic toxico logypractice.

IntroductionInstrumental methods are the cornerstone of modernforensic toxicology analyses. This review presents the theoryand most recent applications of current technology for analysisof toxicants in biological fluids and tissues. The descriptions ofmethodological theory are necessarily brief, and readers whowish additional detail are referred to a more comprehensivetext (1). We discuss methodological limitations and innovations that address these limitat ions. To fully understand instrument performance, one must be familiar with commoncharacteristics of analytical assays. These include signal-tonoise ratio (SIN), limit of detection (LOD), lower limit of quantification (LOQ), upper limit of quan tification (ULOQ),accuracy, precision, interference, and robustness. Because of

the importance of these parameters for characterizing assays,they are integral components of method validation (2).Most analytical instruments convert some property ofan analyte into an electronjc or photometric signal. Many factors,such as random electronic or spurious photon transmissions,fluctuating concentrations of inherent substances at the detector, and others, contribute unwanted signal that may inter- The opinions in rhi$ ;utid to are rhost> ol1he :.uthors and do not necessarily ter1ea tht> viev.

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to drug-testing laboratories_ This review is recommended forreaders who wish more details of LC-MS theory. To evaluateadvantages and limitations of LC-MS detectors, it is necessary to understand ionization techniques.Development of electrospray ionization (ESI), one of themost common LC-MS interfaces, greatly expanded and improved LC-MS applications. This technology allows transportof charged particles from atmospheric pressure to the highvacuum required forMS (Figure 6, see page 9A). Analytes of interest are charged in solution, nebulized, and desolvated. Solvent evaporates from each droplet until surface tension will notsustain the siz.e ("Rayleigh" limit) and the droplet explodesinto many smaller droplets ("Coulombic" explosion). Analyteions that have been freed from solvent are propelled into theMS by an electrostatic field. Ions are fragmented by collisionswith N2 or similar gases introduced into the reaction chamber,which is termed collision-induced dissociation (CID). ESI interfaces permit MS analyses of molecules in the molecu larweight range of drugs of abuse, 50 to 600 Da, or largermolecules, including proteins as large as 232 kDa (55) . Thisionization method makes it possible to place multiple chargeson large molecules yielding lower mass-to-charge ratio ionswithin the range of MS detection. ESI is the ionization methodof choice for polar compounds. The Nobel Prize in Chemistrywas shared by John Fenn in 2002 for inventing and refiningelectrospray ionization techniques (56).One limitation of ESl is its susceptibility to matrix effectsthat can cause unwanted ionization suppression or enhancement {57-59). Matrix effects can vary between specimens affecting the relative abundance of ions in the mass spectrum,potentially producing inaccuracy in quantification. Approachesfor detecting and evaluating LC-MS matrix effects are available(58-62). Suppression of ionization is greater with increasedsolvent in the chamber; therefore, an innovation to address thisproblem simply reduces the amount of mobile phase exitingthe column. Nano LC techniques introduce a 100-fold lessmobile phase to the ESI interface compared to typical LC systems (63). In addition to reducing solvent, reduction of matrixconstituents also diminishes interference. Improvements inanalyte identification and quantification include adjusting mobile phase composition to reduce co-elution of matrix components with target analytes and employing matrix-matchedcalibrators and deuterated internal standards.Another difficulty with LC-MS and LC-MS-MS is adduct formation. Presence of solvent in the ionization chamber introduces Na+, K+, or NI-14+ that may combine with analytemolecules.These adducts create ions with mlz23, 40, or 18Dahigher than expected. Adducts can contain multiple salt ionsand form bridges between ions of differing masses complicating interpretation of mass spectra. Elution solvents thatminimize salt ions, for example, formic acid in ultra purewater, are one remedy (64).Development of searchable libraries for LC- MS andLC-MS-MS remains a problem because fragmentation andresulting spectra differ between instruments. In a review ofLC-MS-MS literature, Jansen et al. (65) found that althoughspectra did not offer expected interinstrument reproducibility,they didhave many similar features . Investigators continued to240

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attack this limitation and to report methods for creating reference libraries (66). In a recent report, better reproducibility wasachieved by standardizing collision energies to 20, 35, or 50 eV(67). ESI-MS-MS spectra were created for 800 compounds,demonstrating that libraries are now feasible for general use.Historically, peak capacity, that is, the number of resolvedpeaks within a defined span of retention time, has been lowerfor LC-MS compared to CC-MS. Although still true, LC-MSpeak capacity has more than doubled with a UPLC that employssolid phase particle diameter < 2 microns and pressures > 34megapascals (5000 psi) (68). Additional practical applicationsfor biological fluid analyses are available (69,70).Atmospheric pressure chemical ionization (APCI) is an alternate LC-MS interface with applications for organic compoundsin the 50 to 800 Da range. APCl nebulizes solvent and analytesin a manner similar to electrospray technology, but also createsplasma with a corona-discharge needle (Figure 7, see page9A). In this plasma, proton transfer reactions occur that add additional charge and fragment analyte molecules. Charged particles are electronically filtered before introduction into theMS. In most applications, matrix effects on ionization are lesscommon than with ESI. Adduct formation and variable fragmentation are problematic, as they are for ESI, but can besuccessfully minimized (59).Another fragmentation technique avai lable in LC- QMS,LC- ITMS, and LC-MS-MS applications is CID. CID occurswhen ionized compounds are accelerated in a fixed area by anelectrical charge and collide with neutral gas molecules (N2, Ar,or He) resulting in fragmentation. CIDcan occur in-source andin the mass analyzer. In an LC-MS system, in-source CIDrefers to fragmentation occurring prior to mass detection. Byapplying a potential difference between the capillary endcap(labeled CID in Figure 6, see page 9A) and the skimmer,molecules are accelerated over a short distance causing themto collide with the drying gas. These collisions result in fragmentation. Increasing the potential difference (fragmentorvoltage) increases rates of collision and produces differing degrees of fragmentation. Voltages are optimized to produce desired fragmentation of each ion of interest. Distance betweenthe endcap and skimmer differs between manufacturers and affects fragmentation of a particular molecule at a fixed voltage.In addition, the weight of the collision gas also affects overallfragmentation. A heavy gas, such as Ar, impacts moleculeswith more energy, causing a greater degree of fragmentation.Also. for some instruments, in-source CIDcan take place in theoctapole region (between the skimmer and MS in Figure 6. seepage 9A) by increasing the applied voltage and increasing thenumber ofcollisions.In tandem quadrupole MS, CID occurs in the second massanalyzer (collision cell, Q2 in Figure 4, see page 8A) by increasing the pressure and accelerating ions to collide with gasmolecules. Similarly, CID can occur inside an ion trap. Energyis applied to the trap, exciting the ions, which then collidewith helium causing fragmentation. Helium also serves tocool and focus the ions inside the center of the trap by forminga buffer between the orbiting ions and the inside walls of thetrap. Mass analyzer CID imparts greater specificity and has ahigher efficiency of collision compared to in-source CID. The

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molecular ions are isolated prior to fragmentation eliminatingthe possibility of co-eluting ion interferences that can occurduring in-source CID. As a result, massanalyzer CID is the preferred method when studying fragmentation patterns and identifying unknowns.As mentioned previously, one advantage of LC-MS is theability to identify and quantify compounds without derivatization. Stanaszek et al. (71) described an LC-APCI-MS procedurethat identified and quantified eight underivatized am phetamines in hair. Assay performance characteristics wereLOD 0.05 ng/mg (50 ppb, 2.5 ng on column), LOQ 0.1-0.3ng/mg (5-15 ng on column), accuracy 16%, and intra- andinterassay concentration variations of less than 12% and 19%,respectively (71). This procedure requires minimal specimenpreparation and has a large linear range.Methods with similarperformance characteristics using LC-ESI-MS are publishedfor eight benzodiazepine metabolites, LSD and metabolites,and several nitrogen-containing drugs (72-74).Miksa et al. (75) demonstrated the advantage of LC- MS foridentifying compounds with differing chemical properties in

the same assay. This APCI method screens for acepromazine,ketamine, medetomidine, and xylazine in 0.3 mL of serum orblood with a 2 ng/mL LOD and relative standard deviation of1-7%. Another LC-MS procedure identified and quantified cocaine, seven metabolites and ecgonidine in blood with an LODof 0.5 ng/mL, 0.5 ng on column (76). Sklerov et al. (77,78) utilized LC-ESI-MS to rapidly identify and quantify drugs andmetabolites in postmortem blood. Examples included identification of overdoses with loperamide (77) and an herbaltryptamine (78). The latter procedure followed a general approach to testing for designer tryptamines in blood and urineusing GC-QMS for screening and LC- ESI-MS for confirmation(64).Analytical methods for quaternary amine neuromuscularblocking agents are needed in forensic toxicology because oftheir involvement in accidental and intentional deaths. Thesecompounds volatilize poorly and are difficult to derivatize,limiting GC applications. Sayer et al. (79) demonstrated the effectiveness of LC-MS in identifying and quantifying sixcommon neuromuscular blocking agents in blood, serum,urine, and gastric contents. Compounds were quaternaryamines with molecular weights in the 500 to 600 Da range,with salts exceeding 1000 Da. An LC-MS-MS me thod for asimilar compound, mivacurium, also has been published (80).Smith (81) reported an LC- APCI-MS method for VX (0-ethyl S-[2-diisopropylaminoethyl] methylphosphonothioate),

an important neurotoxin used as a chemical weapon, demonstrated the usefulness of currently available chiral columns. VXhas two important enantiomers. The isomer with a (-) phosphorus chiral center is an order of magnitude more toxic thanthe(+) isomer. Using a Chiracel OD-H column (Chiral Technologies, Exton, PA), both isomers were separated, identifiedand quantified in human plasma (81).Similar procedures havebeen reported for metabolites ofnicotine in plasma and d,/-dextromethorphan in regulated formulations (82,83).LC-MS-MS applications continue to be developed to handledifficult biological matrices and to improv e specimenthroughput. Herrin et al. (84) examined postmortem blood, a

matrix that typically has many interfering substances. Thepresence of over 400 compounds could be screened using anion trap LC-M3-MS system with LODas low as 5pg/L (5 ng oncolumn). Murphy and Huestis (85,86) developed an assay foranalyzing drugs and glucuronide metabolites in the same analysis; morphine and codeine analytes were quantified in humanurine and buprenorphine and metabolites in human plasma.Fourteen drugs that were substrates for CYP3A4 were identified by Racha et al. (87) using a high throughput LC-MS-MSwith an enzyme inhibition assay for detection. Rule et al. (88)combined high throughput microliter well systems that employed solid-phase extraction prior to LC- MS-MS analysis.Nordgren et al. (89) screened for 23 analytes in urine by directinjection LC-MS and suggested that large volume laboratoriescould replace immunoassays with LC-MS screening to reducefalse-positive test results.There also are other specialty applications for LC-MS-MS. Inaddition to product ion scanning, described in the GC-MS-MSsection, one can scan ions in the first QMS and monitor onlyone ion in the third QMS (Figure 4, see page 8A). This method,termed precursor ion scanning, allows identification in thefirst QMS and quantification using a single ion in the lastQMS. A different application includes capturing spectra ofunidentified synthetic steroids in urine, for which there are noreference standards, followed by monitoring a product ion thatis characteristic of steroid molecules to assist in identification(90). Choo et al. (91) utilized LC-APCI-MS-MS for analyzingdrugs in meconium, a matrix containing many endogenous interferants. Other applications include characterization of glycopeptides and lipids (92-94). Neutral loss scanning is a lesscommon technique but has been helpful in monitoring glucuronides and lipids (93,95) . In the neutral loss scan mode, aspectrum is obtained that shows all the precursor ions that losea neutral moiety of selected mass (Figure 4, see page 8A) . In arecent LC-MS-MS assay, investigators quantified nine metabolites ofcocaine and heroin in a single analysis using selected reaction monitoring (SRM, also called multiple reactionmonitoring) (96,97). SRM methods monitor specific precursorto product ion transitions (Figure 4, see page 8A). The iontransition is relatively unique for a specific compound andmonitoring a single product ion versus multiple fragmentsimproves quantification. Coles et al. (98) reported a similarSRM method that allowed for the simultaneous LC-MS-MSanalysis of six opiates in urine, serum, plasma, blood, andmeconium. Compared to GC-MS, this method increasedspecificity and avoided glucuronide hydrolysis.

A inear or two-dimensional (20) version of the quadrupoleion trap was described in 2002 (99). The quadrupole arrangement of this trap is similar to the QMS described earlier (Figure8, see page 9A). Ions enter the trap through the opening in thefront and traverse back and forth along the z-axis, trapped bya radial oscillating electric field (radio frequency). The ions arepropelled into the trap and their kinetic energy reduced(cooled) by interactions with gas molecules such as helium introduced as was discussed in the GC-ITMS section. When theoperator lowers the trapping potential, that is, the mass-selective instab ility technique, ions are radially ejected with anarrow kinetic energy range, as low as 0.4 eV. Radial ejection241

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is important to reduce unwanted energy transfer from motion along the z-axis. The trapping potential change also can beregulated to produce pulsed j e c t i o n of ions.Acommon instrument for analyzing ejected ions is a time-of-flight (TOF) MS. Asdiscussed later, the nearly monoenergetic ions entering theTOFMS improves mass accuracy. Unlike the 3D-ITMS, the 2DITMS can be continuously filled with ions while other ionsare ejected in pulses from the radial port. Compared to a 30-ITMS, the 20-ITMS has a 15 times higher ion capacity, 3 timesfaster scan rate, 100% versus 50% detection efficiency, and70% versus 5% trapping efficiency (99-101). Mayya et al. (100)compared 20-ITMS with 30-ITMS using over 100,000 MS-MSspectra acquired with identical complex peptide mixtures andcommon acquisition parameters. More than 70% of the doublyand triply charged particles, but not the singly charged particles, had better quality of fragmentation spectra. They concluded that the number of peptides and proteins identifiedincreased four- to sixfold when using the 2D-ITMS.Other LC-MS-MS methods published in the past three yearsinclude opiates and cocaine in oral fluid (102); ricinine inurine (103); sulfur mustard metabolites in serum (104- 106);poly-chlorinated hydrocarbons in plasma (107); nalmafene inplasma (108); butadiene and metabolites in urine (109); risperidone and its 9-hydroxy metabolite in plasma (110); quetiapinein plasma (111); buprenorphine and norbuprenorphine inserum (112); and 26 benzodiazepines and metabolites,zolpidem, and zopiclone in blood, urine, and hair (113). LODfor these methods was about 0.1 ppb (10 to 250 pg on column) .Many of these compounds are typically found in low concentrations and are difficult to analyze by GC methods. Benzodiazepines, zolpidem. and zopiclone are often used by perpetrators in drug-facilitated sexual assault and have low concentrations in blood and urine collected from the victim days afteringestion. Investigators often obtain specimens in this delayedtimeframe and want the longest detection times possible. Inthe last study cited, an LC- MS-MS assay allowed analysis ofmultiple matrices with detection times for urine of greaterthan 14 days (113,114).With low detection limits, hair analysismay be able to detect single doses of some of these drugs andimprove a police investigator's ability to identify single ingestion of a substance used for drug-facilitated sexual assault(115-117).One method to reduce unwanted ionization effects in LC-MSis to reduce specimen volume. Of course, this also reducesanalyte abundance and, therefore, signal strength. New iontrap LC -MS-MS systems with lower detection limits makepossible the detection of small amounts of sample on column.Tomkins et al. (63) reported a chip-based nanoelectrosprayMS-MS, SRM mode, method that could detect 0.49 ng cotinine, a metabolite of nicotine, in 1 mL oral fluid. The chip isthe LC system, and the amount of specimen extract on the chipwas 10 J.lL, providing an LOD of 4.9 pg on column. The assaywas linear to 40 ng/mL, an important feature, because cotininehas a large expected concentration range. This nano LCmethod also employed a 96-well microtiter plate to reducelaborand increase throughput. In this preliminary study, accuracy and precision results were not reported, and it was notedthat despite the small injection volume, occasional specimens242

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had reduced signal that was possiblydue to ion suppressionfrom oral fluid components.The investigators also found thatsome micropipet tips or ESI nozzles were blocked by solidmaterial in the specimen. These problems will need to be resolved by future innovations, but the concept of asystem withsmall specimen size, low detection limits, multiple drug testingplatforms, and high throughput is promising .

Magnetic Sector MS and CC-CombustionIsotope MS

Amagnetic sector MS sorts ions by passing them through achannel in a magnetic field. An ion's route of travel curves inthe constant magneticfield and the arcof the curve is a function of ion mass-to-charge ratio. The detector sorts ions withdifferent mass-to-charge ratios and makes accurate measurements. Using peak matching or dynamic voltage scanningtechniques, ion masses can be measured in I0-3 to 10-s Da increments compared to 10-1 to 10-2 Da increments by typicalQMS or ion trap instruments.The instrument's resolution canexceed 100,000 compared to less than 10,000 for a QMS (resolution = ion mass divided by the smallest measurable massdifference) and mass measurement accuracies of less than 10compared to 100 ppm (118). The more precise molecularmasses provide improved elemental composition yieldingbetter discrimination between unknown substances. Instruments can be expensive but have applications in compoundidentification and sport testing.AGC-isotope combustion MS is a special type of magneticsector instrument that accurately measures the mass of combustion products.As the name implies, the technique separatescompounds and measures the ratios of stable isotopes ofcarbon, nitrogen, and oxygen in combustion products; C02,H2,N2, or CO. Isotope ratios of the original molecules can be calculated from these data. In general, this technique is useful because compounds have different isotope ratios of elementsthat compose them based on origin of precursors . For example, exogenous testosterone has a lower 13CJ12C ratio thanendogenously produced testosterone; this ratio can be used todistinguish testosterone doping in sports (119,120) .The GC separates volatile compounds in the same manner described. Carbon and nitrogen compounds eluting from thecolumn are directed into a combustion reactor with acarriergas, usually He. The compounds are oxidatively combustedwhen passing over a Cu/Ni/Pt wire maintained at greater than900C. Combustion products enter a reduction reactor withCu wires at 600C to reduce nitrogen oxides to nitrogen. Hydrogen and oxygen products are usually prepared in highertemperature reactors.Even though novel applications with thelatte r two elements are reported each year, they are lesscommon in forensic toxicology. After water is removed in aseparator, the abundances of combustion products of differentmasses are measured in a single magnetic sector instrument. Asan example, the abundances of masses 44, 45, and 46 Da aremeasured for the stable isotopes of carbon corresponding toI2CJ60160, 13CJ60I60, and 12CI60I80, respectively. Isotope ratios

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that all molecules are in the same electrochemical state on thereverse voltage ramp. Many biogenic amines have unique reduction potentials and have been analyzed in biological fluidsby cyclic voltammetry (158,159). Modern applications rangefrom screening of antioxidants in pharmaceutical preparationsto in vivo monitoring of dopamine in neuroscience research(160-162).Aproblem with electrode measurements is that the electrodesurface can become fouled, producing interference in currentmeasurements. One solution is aPED, an electrochemical device operating in a mode that applies a pulse program to anoble-metal electrode, producing continuous surface regeneration. PEDs are useful for detecting compounds that are poorchromophores and therefore not easily detected by spectrophotometry. PEDs are not as specific as MS detectors, but muchless expensive. Typical LOD for LC-PED are 100-500 pg oncolumn with concentration intra-assay coefficients of variationin biological fluids of less than 15%. Even though electrodesare not readily fouled, electrode surfaces do eventually deteriorate and need to be changed when analysts observe diminished signal strength of quality control samples. PEDs havebeen used to analyze pharmaceutical preparations with hydroxyl and sulfur-containing organic compounds, explosivecontaining environmental specimens, and enantiomers(163,164). Kaushik et al. (165) identified and quantified ethylglucuronide, a direct metaboli te of ethanol used to assessdrinking status, in human urine; the authors proposed a generalized approach using PED assays to analyze drug glucuronides (166). As part of a study comparing variouselectrodes for PED, Cataldi et al. (167) reported a method foridentifying iodide in urine, a test useful for proving adulterat ion in drug-testing programs.

Capillary ElectrophoresisCE is experiencing a resurgence in toxicology laboratoriesbecause it offers some advantages over chromatographicme thods (168,169). The instrument separates compoundsbased on electrophoretic mobility and interaction with a liquidor solid phase adsorbent instead of the chromatographic mechanism of phase solubility. The basic design of the instrument isa fused silica capillary connected to buffer at one end and normally the same or similar buffer at the other end. Ahigh electric potential difference is placed across the tube creating

electroosmotic flow of buffer as shown in Figure 10. Analytes(neutral compounds, anions, and cations) are introduced intothe tube at the source end and flow with the buffer at differentrates toward the detector, usually the cathode. Although anionsare attracted to the anode, they move toward the cathode dueto the greater magnitude of electroosmotic flow. Other factorsthat influence migration are the number of charges, size, andmass of the analyte. CE offers advantages includ ing requiremen t of only small volumes of buffer and specimen, analysis ofbasic, neutral, and acidic compounds in the same assay, readydetermination of enantiomeric purity, and the capability toanalyze large, water-soluble biomolecules (170). Unlike HPLC,246

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there is no transfer of analyte molecules between mobile andstationary phases, increasing the theoretical plates for CE. Asanalyte migrates, it also is compacted by the same force thatcaused electroosmotic flow.The result is narrow, discrete bandsof analyte at the destination end of the capillary, the location ofthe detector. Detectors include UV, visible, and fluorescencespectrophotometers; PED; and MS.Types of MS and interfacesare similar to those used in LC. Many CE performance characteristics depend upon the detector selected.Reviews of CE have been published in many fields of scienceincluding forensic toxicology (168,171- 173). CE methods havebeen utilized for identifying and quantifying a wide variety ofdrugs and biological compounds (174--176). Lurie et al. (169)reported application of CE for analysis of a variety of controlled substances in a single injection that separated acidic,neutral, and basic drugs. The instrument was inexpensive,simple in design, easy to maintain, and used very smallamounts of specimen and buffer. Sensitivity of CE varies overa general range of milligrams per liter (parts per million) fordetectors such as direct UV absorption to micrograms per liter(parts per billion) for conductivity and laser-induced fluorescence detectors (176,177). Derivatization and sample concentration enhanced sensitivity (172,177-181) . Chiral separationsusing CE methods also have been reported (170-173,178,180,181). Unlike LC or GC methods,which require specializedcolumns, CE chiral separations can utilize more cost effectiveadditives, such as cyclodextrins, to the mobile phase (182).Additives enhance separation depending upon the analytes'distribution coefficients with aparticular cyclodextrin. Innovations in dynamically coated capillaries promise to improveresolution of many different compounds and expand applications, such as accurate determination of pKavalues for drugs(169,183,184). MS detectors improved certainty in identifica-

Cathode(-) 0 a3: 0i' 0 EOF

0. a0 0 0

FIXED MOBILEAnode(+)

Figure 10.Schematicofacapil lary electrophoresis nstrument. Cations,anions andneutral molecules arepropeled towa rd thecathode byelectroosmotic How (EOF} of the buffer.They are separaed based on electrophoretic mobility and nteractionwith the adsorbent beforeenteringa detector positionedat thecathodeendof the capillary.

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tion (173,185). The small amount of compound exiting thecapillary is one limitation of a CE-QMS (ppm LOD) comparedto an LC-QMS (ppb LOD) . Despite this limitation, microchiptechnology using CE is becoming more common (186), andrecent sample enrichment methods for CE-MS have loweredlimits of detection (187). Precision in CE migration times canbe variable under some conditions and may be reduced by mobile phase additives (188) .

Conclusions1\vo Nobel prizes were awarded for novel MS technologieswith applications in forensic toxicology: one to Wolfgang Paulfor the quadrupole ion trap mass spectrometer and another toJohn Fenn for electrospray ionization methods. The QMS,ITMS, and LC-ESI-MS are important instruments derived fromthese innovations. With these tools, toxicologists routinelyidentify and quantify a range of compounds in biological fluids

from small molecular weight drugs, steroids, and toxins to232 kDa macromolecules .Recent refinements such .as better vacuum pumps and novelion sources for GC-MS have improved ruggedness, a necessaryrequirement for analyzing biological specimens such as blood,hair, and soft tissues. Some GC-QMS instruments, for example, are still operating in high-volume laboratories aftertwo decades of use.Innovations have reduced the time required for analysis.Examples include reduced column bore size, high heating rateovens, high pressure carrier gas control, and more efficientcapillary columns leading to fast GC methods. Software designhas made LC-MS and LG-MS-MS operation easier, faster, andwithin the abilities of less experienced laboratory analysts.Thedevelopment of ESI and APCI interfaces expanded LC-MS applications and improved reliability of quantification. New additives to mobile phases and dynamic coatings improvedresolution and precision in CE. Some laboratories have transitioned from chromatography to CE methods to reduce retention times and analyze mixtures for acid, neutral and basiccompounds in a single run. The 20-ITMS with monoenergeticexpulsion of ions combined with better ion reflectors in TOFMS have resulted in greater mass accuracy measurements.These modern instruments identify compounds by massspectra that are collected in seconds compared to minutes formore traditional MS systems. One promising application isidentification of unknown compounds for which there are noreference standards using exact mass measurements.The quest to achieve lower limits of detection led to developments in both chromatography and MS. The Deans Switch, atwo-dimensional chromatographic technique, allows analyststo select eluents from aGC column in small segments of time.This reduces interferences from unwanted compounds elutingjust prior to the retention time of interest and also preservesthe MS detector. Modern MS-MS, after two decades of improvement, are smaller, more rugged, and have more userfriendly software compared to predecessors. Theseimprovements have made MS-MS more useful in forensic tox-

icology laboratories, especially when methods requiring anLOD of less than 1pg!L (I ng on column) are needed, such aswith hair, oral fluid, and sweat testing analyses. Of course, theultimate instrument for a low LOD, as well as mass accuracyand resolution, is the FTMS. One publication documented anLOD of nine attomoles, or 0.26 pg (139) . The instrument hasgreater sensitivity for multiply charged ions making it ideal forexamining macromolecules.Hybrid instruments are one tool to help analysts addressthe expanding requirements of forensic toxicology laboratoriesin criminal investigations. An LC-2D-IT-TOF-MS linked to aFTMS allowed investigators to collect identifying informationfrom CID experiments in the first MS and then capture, on-demand, exact mass information for selected ions in the FTMS.Applications for this type ofversatile instrumentare promising.Improvement of LC interfaces, columns, flow rate pumps, andoperating soft\vare have made LC an option for non-researchforensic toxicology laboratories. Now with acceptable precision,resolution, and quantification, analysts can exploit the advantages of an LC- MS over a GC-.MS by examining biologicalfluids for nonvolatile compounds. The quest to routinely exam ine drugs and glucuronide metabolites in the same analysismay be realized (189).Although achievements in analytical toxicology are many,limitations remain. LC-MS spectra are not reproducible between instruments making it difficult to compile commonspectral libraries. Standardization of collision energies isaddressing this limitation, but compilation of spectral librariesis in its infancy. Ion suppression remains a problem forsome matrices and applications. To achieve peak capacityequal to GC- MS, higher pressures are needed and some recentcommercial instruments and columns routinely achieve 103megapascals (15,000 psi). For equivalency in separation ofanalytes, however, Medina et al. (68) calculated that about 620megapascals (90,000 psi) would be needed. This high pressuremay not be practical. Another solution would be new innovations in columns, mobi le phases and high temperaturemethods that could enhance separation. Microchip methodsshow promise but practical problems with clogging of narrowchannels and sporadic ion suppression limit expansion toroutine testing.The need in CE applications is amore sensitivedetector. There are a number of methods published for CE-MS,but more sensitive and robust procedures are needed beforethis instrument can reach its potential in forensic toxicologylaboratories.The biggest limitations for FTMS are its high cost and requirement of ultra low vacuum. The Orbitrap, a new technology introd uced in 2002, may remedy some of the problemsby measuring accurate masses at a lower cost and with fewermaintenance requirements .The authors hope that colleagues will pursue and discovernew innovations to overcome limitations of our current analytical methods and that these innovations will be applied to theanalysis of an expanded inventory of analytes and matrices inforensic toxicology laboratories.Improving instrument ruggedness, increasing throughput, lowering LOD and LOQ, and developing versatile systems to expand applications will help usmeet future analytical challenges.

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27. T. Gunnar, K. Ariniemi, and P. lillsunde. Validated toxicologicaldetermination of 30 drugs of abuse as optimized derivatives inoral fluid by long column fast gas chroma tography/electron impact mass spectrometry. /. MassSpectrom. 40: 739- 753 (2005).28. T. Gun nar, K. Ariniem i, and P. Lillsunde. Fas t gas chromatography-negative -ion chem ical ionization mass spectrometry withmicroscale volume sample preparation for thedetermination ofbenzodiazepines and alpha-hydroxy metabolites, zaleplon andzopiclone in whole blood. f. Mass Spectrom. 41: 741-754(2006).29 . T. Gunnar, T. Eskola, and P. Lillsunde. Fast gas chromatography/mass spectrometric assay for the validated quantitative determination of methadone and the primary metabo li te EDDP inwhole blood. Rapid Commun. Mass Spectrom. 20: 673-679(2006).30. L.D. Bowers. Analytica l advances in de tection of performance enhancing compou nds. Clin. Chem. 43: 1299- 1304 (1997).31. Y.L. Tseng, F.H. Kuo, and K.H. Sun. Quantification and profi l ing of 19-norandrosterone and 19 -noretiocholanolone inhuman urine after consumpt ion of anu tritional supplement andnorsteroids. }. Anal. Toxicol. 29: 124-134 (2005).32 . C. Moore, S. Rana, C. Cou lter, F. Feyerherm, and H. Prest. Application of two-dimens ional gas chromatography with electron capture chemical ionization mass spectrometry to thedetection of 11 -nor-?9-tetrahydrocannabinol-9-carboxylic acid(THC-COOHl in hair. }. Anal. Toxicol. 30: 171-1 77 (2006).33. D. Day, D.j. Kuntz, M. Feldman, and L. Presley. Detection ofTHCA in oral flu id by GC-MS-MS. }. Anal. Toxicol30: 6 4 5 ~ 5 0(2006).34. M. Dunn, R. Shellie, PMorrison, and P. Marriott. Rapid sequential heart-cu t multidimensional gas chromatographic analysis.}. Chromatogr. A 1056: 163- 169 (2004).35. S.M . Song, PMarriott, A. Kotsos, O.H. Drummer, and P. Wynne.Comprehensive two-dimensional gas chromatography withtime -of-fligh t mass spectrometry (GC x GC- TOFMSl for drugscreening and confirmation. Forensic Sci. Int. 143: 87-101(2004).36. j. Harynuk and P.). Marriott. Fast GC x GC with short primarycolumns. Anal. Chem. 78: 2028-2034 (2006).37. M. Wang, P.J. Marriott, W. H. Chan, A.W. Lee, and C.W. Huie.Enantiomeric separation and quantification ofephedrine-type alkaloids in herba l materials by comprehensive two -dimensionalgas chromatography.]. Chromarogr. A 1112: 361 - 368 (2006).38. D. Ryan, PMorrison, and P. Marriott. Orthogonality considerations in comprehensive two-dimensional gas chromatography.}. Chromatogr. A 1071: 47 -53 (2005).39. X. Lu, H. Kong. H. Li, C. Ma, ). Tian, and G. Xu. Resolution prediction and optim ization of emperature programme in comprehensive two-dimensional gas chromatography.]. Chromatogr. A1086: 175-1 84 (2005).40. R.D. Scurlock, G.B. Ohlson, and D.K. Worthen. The detectionof t.9-tetrahydrocannabinol (TH Cl and 11 -nor-9-carboxy-119-tetrahydrocannabinol (THCA) in whole blood using twodimensional gas chromatography and El-mass spectrometry.f. Anal. Toxicol. 30: 262- 266 (2006) .

41. F.W. Mclafferty. Tandem mass spectrometry. Science 214:280- 287 (1981 .42 . l . Politi, A. Groppi, and A. Polettini . App lications of liquidchromatography-mass spectrometry in dop ing contro l. }. Anal.Toxicol. 29: 1- 14 (2005}.43. S.A. Reuschel, D. Eades, and R.L. Foltz. Recent advances inchromatographic and mass spectrometric methods for determination oi LSD and its metabolites in physio logical specimens.f. Chromatogr. 8733:145- 159 (1999).44 . ).R. Barr, W.J. Driskell, L.S. Aston, and R.A. Martinez. Quantitation of metabolites of the nerve agents sarin, soman, cyclohexylsarin, VX, and Russian VX in human urine usingisotope-dilu tion gas chromatography-tandem mass spectrometry. f.Anal. Toxicol. 28: 372-378 (2004).

45. A.E. Boyer, D. Ash, D.B. Barr, C.L. Young, W.J. Driskell, R.D.Whitehead, Jr., M. Ospina, K.E. Preston, A.R. Woolfitt, R.A.Martinez, L.A. Silks, and j.R. Barr. Quantitation of the sulfur mustard metabo li tes 1, 1'-sul fon ylbis[2 -(methylthio}ethane] andthiodiglycol in u ri ne using isotope-dilution gas chroma tography- tandem mass spectrometry. }. Anal. Toxicol. 28: 327- 332(2004).46. E.M. Jakubowski, J.M. McGuire, R.A. Evans, j.L. Edwards, S.W.Hulet, B.). Benton, j.S. Forster, D.C. Burnett, W.T. Muse, K.Matson, C.L. Crouse, R.J. Mioduszewski, and S.A. Thomson.Quantitation of fluoride ion released sarin in red blood cellsamples by gas chromatography--

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joumal of Analytica l Toxicology, Vo l. 31, June 2007of biogenic am ine neurotransmission in rea l t ime. /. Pharm.Biomed. Anal. 19: 33-46 (1999).163. 5.). Modi, W.R. LaCourse, and R.E. 5hansky. Determinatio n ofthio-based additives for biopharmaceuticals by pulsed electrochemical detection following HPLC.). Pharm. Biomed. Anal. 37:19-25 (2005).164. W.R. LaCourse. Pulsed Electrochemical Detection in High-Performance Liquid Chromatography. Wiley, Indianapolis, IN,1997, p 352.165. R. Kaushik, W.R. LaCourse, and B. Levine. Determination ofethyl glucuronide in urine using reversed-phase HPLC andpulsed electrochemica l detection (Part tl). Anal. Chim. Acta556: 267- 274 (2006).166. R. Kaushik, B. Levine, and W.R. LaCourse.A brief review: HPLCmethods to directly detectdrug glucuron ides in biolog ica l matrices (Part I). Anal. Chim. Acta 556: 255-266 (2006).167. T.R. Cata ldi,A. Rubino, M.C. Laviola, and R. Ciriello. Compa rison of silver, go ld and modified platinu m electrodes for theelectrochemica l de tection of iodide in urine samples followingion chromatography. /. Chromatogr. 8 827: 224-231 (2005).168. W. Thormann. Progress of capillary electrophoresis in therapeutic drug monitoring and cl inica l and forensic toxi co logy.Ther. Drug Monit. 24: 222- 231 (2002).169. I.S. Lur ie, P.A. Hays, and K. Parker. Cap illary electrophoresisanalysis of a wide var iety of seized drugs using thesame capi llary with dynamic coa tings. Electrophoresis 25: 1580-1591(2004).170. U. Holzgrabe, D. Brinz, S. Kopec, C. Weber, and Y. Bitar. Whynot using capillary electrophoresis in drug analysis? Electrophoresis 27: 2283-2292 (2006).171. S. Zaugg and W. Thormann. Enantioselective determination ofdrugs in body Ouids by capillary electrophoresis.). Chromatogr.A 875: 27-41 (2000).172. 5. Fanali. Enantioselective determination by capillary electrophoresis with cyclodextrins as chira l selectors.). Chromatogr.A 875: 89-122 (2000).173. 5.A. Shamsi. Ch iral capil lary electrophoresis-massspectrometry:modes and appl ications. Eectrophoresis 23: ~ (2002).174. C.M. Boone, JW. Do uma, J.P. Franke, R.A. de Zeeuw, andK. Ensing. Screening for the presence of drugs in serum andurin e using different separat ion modes of ca pillary elec trophoresis. Forensic Sci. Int. 121: 89-96 (2001).175. S. Chinaka, 5. Tanaka, N. Takayama, N. Tsuji, S. Takou, andK. Ueda. High-sens itivity analysis of cyanide by capillary electrophoresiswith fluo rescence detection. Anal. Sci. 17: 649-652(2001).176. M. Frost and H. Kohler. Ana lysis of lysergic acid diethy lam ide:compa ri son of capillary electrophoresis with laser-inducedfluorescence !CE-LIF) with conventional techn iqu es. ForensicSci. Int. 92: 213-218 (1998).177. C. Huhn, M. Putz, N. Ma rtin, R. Dah lenburg, and U. Pyel l. Determ ination of tryptamine der ivatives in illicit syntheticdrugs bycapillary electrophoresis and ultraviolet laser-induced fluorescence detection. Electrophoresis 26: 2391 - 2401 (2005).178. R. lio, S. Chinaka, N. Takayama, and K. Hayakawa. Simulta-

neous ch ira l ana lysis of methamphetamine and re lated compounds by capi llary electrophoresis/mass spectrometry usinganionic cyclodextrin. Anal. Sci. 21: 15- 19 (2005).179. W.j. Underberg and ).C. Waterval. Derivatization trends in capillary electrophoresis: an update. Electrophoresis 23: 3922- 3933(2002).180. N. Pizarro, ). Ortuno, M. Farre, C. Hernandez-Lopez,M. Pujadas, A. Llebaria, ). Joglar, P.N. Roset, M. Mas,). Segura,). Cami, and R. de Ia Torre. Determ ination of MDMA and itsmetabolites in blood and urine by gas chromatography-massspectrometry and analysis of enan tiomers by capillary electrophoresis. f. Anal. Toxicol. 26: 157-165 (200 2).181. 5. Rudaz, L. Geiser, 5. 5ouvera in, ). Prat, and ).L. Veuthey. Rapidstereoselect ive separat ions of amphetami ne deri vatives withhighly sulfa ted gamma-cyclodex trin. Eectrophoresis 26:3910-3920 (200S).182. T. Ramstad. Enantiome ric purity methods for three pharmaceutica l compounds by electrokinetic capillary chromatographyut ili zing highly sulfated-gamm a-cyclodextrin as the chiralselector. }. Chromatogr. A 1127: 286-294 (2006).183. L. Geiser, Y. Henchoz, A. Ga ll and, PA. Ca rrupt, and).l. Veuthey. Determination of pKa values by capillary zoneelectrophoresis with a dynamic coating procedure. }. Sep. Sci.28: 2374-2380 (2005).

184. K.W. Phinney and L.C. Sander. Dynamica lly coated capi llariesfor enantioselective separations by capi llary electrophoresis.Chirality 17 Suppl: 565-569 (2005).185. E.M. Weissinger, B. Hertenste in, H. Mischak, and A. Ganser. Online coupling of cap illary electrophoresis with mass spectrom etry for the identification of biomarkers for clinical diagnosis.Expert Rev. Proteomics 2: 639-647 (2005).186. C.S. Henry. Microchip capillary electrophoresis: an introdudion.Methods Mol. Bioi. 339: 1-10 (2006).187. K. Sandra, F. Lynen, B. Devreese, ). Van Beeumen, and PSandra.On -co lumn sample enrichment for the high-sensitivity sheathnow CE-MS analysis of pep tides. Anal. Bioanal. Chern. 385:671-677 (2006).188. Z. Dey I, I. Miksik, and F. Tagliaro . Advances n cap illary electrophoresis. Forensic Sci. Int. 92: 89- 124 (1998).189. M. ). Bogusz, R.D. Ma ier, M. Erkens, and S. Dri essen. Determi nation of morphine and its 3- and 6-glucuronides, codei ne,codeine-glucuronide and 6-monoacetylmorphine in body flu idsby liquid chromatography atmospheric pressure chemical ionization mass spectrometry. }. Chromatogr. B 703: 115- 127(1997).Manuscript received December 29, 200 6;revision rece ived March 27, 2007 .

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Modern Instrumental Methods in ForensicToxicology rsee PP 237-253)Detector

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Figure 1. Schematic showing resonant ons of selected mass-to-chage ratios being sorted anddetected in a quadrupole mass spectrometer(QMS).A constant DC to AC current ratio (AC cu rre nt prod ucesa radiofrequency field) selects the resonant ion.

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Q ' ""--ar-' ll3Figure 4. Schematic showing a andem mass spectrometer (MS-MS). Ina linear quadrupole instrument, Ql, Q2, and Q3 represent the threetandem quadrupoles. Four different operational configurations areshown: product ion scann ing provides structural information (A); precursor ion scanning identilles all compounds producing specific fragments (8); neutral loss scann ing screens for compounds producingneutral loss ragments (e.g., m/z 176 ior glucuronide conjugates) (C); andselected reaction monitoring monitors targeted analytes and simultaneouslymonitorsmultiple ransitions (D). Source: Thermo FisherScienrific.

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ElectrodeFigure 5. Schematic of ions sorted in an ion trap mass spectrometer(ITMS). Ions are accumulated, expelled on demand, and detected.

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charged o p l e t ~.LC Spray needle tip \

charged "Co' 1 b' "Spray needl.e tip u om tcdroplets explosion I' ...-.... - ~. . . . . . + ... .....,........ ~ ......-!'. ~ ~ , : :solvent + ; I~ u evaporation \ I analyte "Rayleigh" yteI molecule. limit is reached 1005!L +v -vfigure 6. Schematic of an electrospray ioniza tion (ESI) interface. Chargeddropletsexplode during drying creating charged analytes hat are ilteredprior to entering amassanalyzer. Additional analytemolecules ionizefromcollison-induced dissociation (CIDl with N2 molecules.

corona discharge needle

Figure 7. Schematic of an atmospheric pressure chemical ion ization(APCI) nterface. Solvent droplets. areevaporated prior to heana lytecontacting acorona discharge needle. Theneedlecrea tesprimary and secondary ions ha t transfer charge to analyte molecules during collisons.Additional fragmentation occurs from coll ision-induced dissociation(CID)with N2 molecules.

ylzX FrontSection Radial Ejection Slots:30mm x .25mmfigure 8. Schematic of a wo-dimensional linear on rap mass spectrometer (20-ITMS). Ions traverse back and forth along the z-axis, trapped bya radial oscillating eectric field (radio frequency). Thekinetic energiesof the ions are reduced (cooled) by interactions with gas moleculessuch as helium ntroduced separately. Ions arerad ially eected when thetrapping potentiais owered and strike an ion detector.

Source: ThennoFisher Scientific

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- - - - - - - - - - ~ ~ ~ - - - - - - - - - - - - - - - - ~Figure 9. Schematic of a Penning trap, the central component of aFourier transform ion cyclotron resonance mass spectrometer (FTMS).Ionsenter the box, are rapped in the magnetic ied, excited to larger, coherent orbital radii to facilitatedetection, and the frequency of current nduced in detection pla tes is measu red and transform ed to amass-to-charge ratio.