GAS CHROMATOGRAPHY AND MASS SPECTROMETRY IN...

GAS CHROMATOGRAPHY AND MASS SPECTROMETRY IN CLINICAL CHEMISTRY 1

Gas Chromatography andMass Spectrometry in ClinicalChemistry

Roger L. BertholfUniversity of Florida Health Science Center,Jacksonville, USA

1 Introduction 1

2 Instrument Design and Theory 22.1 Gas Chromatography 22.2 Mass Spectrometry 32.3 Combined Gas Chromatography and

Mass Spectrometry 4

3 Specimen Preparation 43.1 Considerations for Biological

Specimens 53.2 Preparation of Volatile Derivatives

for Gas Chromatography 53.3 Solid Sampling Techniques for Mass

Spectrometry 5

4 Applications to Drug Measurements 64.1 Drug Screening by Gas

Chromatography 64.2 Quantitative Applications of Gas

Chromatography/Mass Spectrometry 8

5 Clinical Applications of MassSpectrometry 135.1 Screening for Inborn Errors of

Metabolism 135.2 Protein Measurement 145.3 Drug Measurements 165.4 Elemental Analytes 175.5 Characterization of Protein Epitopes 175.6 Detection of Genetic Mutations 17

Abbreviations and Acronyms 18

Related Articles 18

References 19

Automated spectrophotometric, electrochemical, and im-munochemical analyses have become the mainstays ofclinical chemistry and toxicology laboratory services,but their scope is limited. A wide array of clinicallyrelevant analytes demand more sophisticated analyticaltechniques to provide sensitive and specific assays for

diagnostic purposes. Gas chromatography (GC) andmass spectrometry (MS) are becoming cost-effectivealternatives for many of these challenging assays. GCis a robust technique that offers the ability to resolvevolatile components of a complex mixture. MS providesstructural information that can unambiguously identify acompound. In combination, these two techniques providequalitative and quantitative answers to many difficultanalytical problems. GC and MS applications have beendeveloped for a variety of clinical analytes, and the use ofthese methods has created new and exciting frontiers forclinical laboratory medicine. Several clinical applicationsof these analytical techniques will be discussed in thischapter.

1 INTRODUCTION

GC and MS are complementary techniques that togethercreate a powerful and versatile analytical method. Sepa-ration of the volatile components of a mixture by GC is atechnology that was first described in 1952,.1/ and it wasimmediately recognized as an indispensable tool for theanalysis of organic compounds. Of particular importancein the evolution of GC toward modern instruments wasthe introduction of capillary chromatographic columns,which improved the resolution of GC separations byseveral orders of magnitude. There are two significant lim-itations of GC as a qualitative and quantitative analyticaltechnique, however. The first limitation is the necessity foranalytes to be sufficiently volatile and thermally stable tovaporize at practical temperatures. A second limitation isthe specificity of GC detectors, which can range from verynonspecific (e.g. thermal conductivity, flame ionizationdetectors (FIDs)), to highly specific (mass spectrometer).GC/MS combines the resolving capabilities of GC withthe unique structural information from MS, making it thehybrid analytical method of choice for qualitative analy-sis of suitably volatile organic compounds. Quantitativeapplications of GC/MS are more complicated, and typi-cally require internal standards. The ability to resolve thecomponents of complex mixtures, and provide qualitativeinformation about organic molecules, makes GC/MS anattractive technique for biomedical applications. Severaldefinitive methods for bioanalytes are based on GC/MSapplications. A common application of GC/MS in clin-ical laboratory operations is for toxicological purposes,including identification and measurement of drugs ofabuse in urine.

As an independent analytical method, GC is also usefulin clinical chemistry and toxicology laboratories. Themethod can be used to screen for a wide variety ofdrugs and metabolites in urine, where chromatographic

Encyclopedia of Analytical ChemistryR.A. Meyers (Ed.) Copyright John Wiley & Sons Ltd

2 CLINICAL CHEMISTRY

retention times are used for presumptive identificationof the compounds detected by flame ionization ornitrogen–phosphorus techniques.

MS has limited standalone applications, since spec-imen purity is essential. MS methods for measuringlow-boiling compounds require a procedure that willvolatilize enough molecules to be detected. There areseveral approaches to MS measurement of nonvolatilecompounds, including liquid chromatography/MS inter-faces, fast atom bombardment (FAB), electrospray, ther-mospray, and matrix-assisted laser desorption/ionization(MALDI). All of these methods incorporate techniquesthat ultimately produce vapor-phase molecules that aresubsequently fragmented in the mass spectrometer’s ionsource.

2 INSTRUMENT DESIGN AND THEORY

2.1 Gas Chromatography

A typical gas chromatograph comprises three fundamen-tal components: an injection system, a chromatographiccolumn, and a detector. In most cases, specimens for GCanalyses are dissolved in a volatile solvent, although neator gaseous specimens can also be used. Most GC injectionsystems are designed to vaporize liquid specimens, andthey accomplish this by heating the injector body to atemperature above the boiling point of the solvent andanalyte. In older GC designs, the sample was injecteddirectly into the chromatographic column, which waspreheated. However, the introduction of capillary chro-matographic columns, which have bores half a millimeteror less in diameter, required innovative injector designs.The challenge was to avoid peak broadening due to leak-age of residual sample into the capillary column overan extended period of time. One microliter of specimen,when volatized, occupies a considerable volume withinthe injector body, and the small caliber of the capillarycolumn cannot accommodate the large volume of vapor.One approach to minimizing the injection bandwidth isto constantly purge the injector body so that only a smallamount of the vapor has the opportunity to enter thecapillary column – this technique is called split injection.The split ratio (amount of specimen entering the columnversus the amount purged) typically varies from 1 : 10 to1 : 99. A limitation of split injection is the loss of analyticalsensitivity, since a smaller amount of specimen enters thecolumn and detector. In some cases, the loss of analyticalsensitivity is not problematic, and may even be beneficial,especially when analyte concentration is high and thedetector’s range of linear response is limited.

Another approach to capillary column injectors issplitless, or Grob, injection, in honor of the technique’s

inventor. In a splitless injection, the injector body iskept hot enough to vaporize the specimen and solvent,but the column temperature remains below the boilingpoint of the solvent. As the vaporized specimen entersthe capillary column, it condenses and therefore thebandwidth is minimized. After a sufficient period of time(usually about 60 s), the injector body is purged andthe column is warmed up to re-vaporize the specimenand begin the chromatography. Splitless injections aretechnically more complex and involve more variablesthan split injections, but a significantly greater amount ofspecimen is delivered to the capillary column, resulting inbetter analytical sensitivity.

On-column injections with capillary columns are alsopossible, and require specially designed syringes fittedwith needles that terminate with a length of very smallcapillary, which fits inside the chromatographic column.Because of the fine capillary point, the syringes aredelicate, and generally not compatible with autosamplermechanisms.

For sufficiently volatile compounds, vapor may beinjected into the gas chromatograph using an airtightsyringe. Raoult’s law states that the mole fractionscontained in the vapor phase above a liquid aredetermined by the respective vapor pressures of theconstituents of the liquid, which in turn are proportionalto their relative concentrations. Therefore, the vaporin equilibrium with a liquid can be used to quantifyvolatile constituents in the liquid – this technique is calledheadspace analysis. Headspace sampling offers severaladvantages over conventional liquid injections: the vaporis substantially free of nonvolatile constituents that mayform residue inside the injector; the injection bandwidthis considerably reduced; and specimen delivery is morenearly quantitative. Headspace analysis is only useful forhighly volatile compounds such as low-molecular-weightalcohols.

GC column performance improved dramatically withthe introduction of fused-silica capillary columns, a tech-nology derived from fiber optics. Resolution equivalent toseveral hundred thousand theoretical plates is commonlyachievable with capillary GC columns. Microprocessorcontrol of the GC oven temperature has enhanced theability to program temperature changes, improving boththe resolution and speed of GC analyses. In most GCcolumns the stationary phase is a liquid and the ana-lytical method is therefore gas–liquid chromatography,following the widely used convention of specifying thestate of both stationary and mobile phases in the namesof chromatographic applications. Gas–solid chromato-graphy applications also exist, but are less common. Theliquid stationary phase may be coated on a solid supportor chemically bonded to the inner wall of a fused silicacapillary column (‘‘bonded phase’’ columns).


The choice of GC detector depends on the type ofcompound that is to be measured, the sensitivity that isrequired, and the degree of selectivity necessary to avoidsignificant interference. Thermal conductivity detectorshave moderate sensitivity, but are not selective. FIDs havebetter sensitivity, and respond mostly to hydrocarboncompounds. Nitrogen–phosphorus detectors are specificfor nitrogen- and phosphorus-containing compounds, andare very sensitive. Electron capture detectors can measurechlorine-containing compounds in subpicogram amounts.The properties and performance characteristics of variousGC detectors are summarized in Table 1.

The versatility and ruggedness of GC makes this analyt-ical method an attractive choice for the measurement ofeasily vaporized compounds, which include many drugs,steroid hormones, vitamins, and metabolic products. Thefactor that has most limited the incorporation of GC intoroutine clinical laboratory services is the low throughputand difficulty in automating GC applications.

2.2 Mass Spectrometry

Several instrumental techniques have been devised toseparate and measure charged particles based on theirmass. A typical mass spectrometer consists of fourcomponents: an inlet system, an ion source, a massanalyzer, and a detector. The first of these components,the inlet system, must ensure that a pure compoundis delivered to the mass analyzer. For this reason,chromatographic systems are a popular choice for amass spectrometer inlet system. The ion source is wherethe compound is ionized, a process that is ordinarilyfollowed by decomposition of the analyte into unique,charged fragments. The mass analyzer sorts the chargedfragments and the detector measures the number ofcharged fragments of any given mass.

Since a mass spectrum (sometimes called a mass frag-mentogram) uniquely identifies a compound based on itsfragmentation pattern, superimposition of the fragmentsfrom a second compound in the ion source would makethe spectrum ambiguous. Therefore, the inlet system fora mass spectrometer must deliver pure compound to theion source in order for the mass spectrometer to be useful

Table 1 Performance characteristics of common GC detectors

Detector Detection Linear Applicationlimit range

Thermal conductivity 0.5 ng 105 UniversalFlame ionization 10 pg 107 HydrocarbonsElectron capture 0.05 pg 104 HalidesThermionic (nitrogen– 0.1 pg 103 N, P

phosphorus)Mass spectrometer 10 pg 106 Universal

for qualitative analysis. Inlet systems for MS include GC,liquid chromatographs, and several methods for vapor-ization and ionization of nonvolatile compounds. The ionsource in a mass spectrometer usually operates under avacuum – the presence of oxygen and nitrogen may affectionization and contribute interfering fragments to themass spectrum – so a pressure differential exists betweenthe ion source and inlet system. This pressure differentialis difficult to maintain when the inlet system is pres-surized, as are gas and liquid chromatographs. Severaldevices have been created to remove the mobile phaseas it elutes from the chromatographic system so that onlyanalyte enters the ion source; examples are vacuum jetseparators (for packed-column GC systems), and moving-belt solvent evaporators (for high-performance liquidchromatographs). Capillary GC columns can usually ter-minate at the entrance to the ion source since the minimalcarrier gas flow can be removed efficiently by the massspectrometer’s vacuum system.

When solid sampling systems for nonvolatile analytesare used, the pressure differential is less of a concernbecause the sampling system can operate under vacuum.Solid sampling inlet systems include MALDI, FAB,thermospray, and electrospray. In a MALDI system, theanalyte is embedded into a pure crystalline matrix. Whena laser is directed at the crystal, analyte and crystalmolecules are ejected. FAB is a similar technique, exceptthat high-energy beams of inert atoms, such as argon,are used to initiate molecular ejection. In electrosprayionization, the analyte is dissolved in an organic solvent,and passed through an electrically charged capillary.Small clusters of analyte/solvent form in the capillary, andbecome charged. As the clusters are accelerated througha series of lenses, the solvent is gradually removed,resulting in smaller and smaller clusters. When the clustersreach a certain size, coulombic forces cause them toexplode, and the resulting fragments are measured inthe mass analyzer. Thermospray ionization is a similartechnique, except that the capillary is heated, and solventevaporates quickly after the analyte/solvent aerosol exitsthe capillary. In both electrospray and thermosprayapplications, nonvolatile analytes are stranded in thevapor phase as solvent is removed, and can thereforeenter the mass analyzer and be measured. These solidsampling techniques are particularly useful for high-molecular-weight compounds, which include proteins andnucleic acids.

The ion source of a mass spectrometer shatters the ana-lyte molecules so that their fragments can be separatedand measured. Most mass spectrometers use a high-energy flux of electrons to ionize molecules – the methodis called electron impact ionization. Most reference massspectra are generated by electron impact ionization.There are circumstances, though, when electron impact


ionization does not produce satisfactory spectral unique-ness or analytical sensitivity, and other ionization methodsmay be preferable. One alternative method is chemi-cal ionization, in which the ion source is pressurizedwith a reagent gas. The electron flux ionizes the reagentgas, which in turn interacts with the analyte to producecharged species. This approach is particularly useful forgenerating negatively charged ions. Fragments may alsobe produced by collisional dissociation, where analytemolecules (or fragments) are accelerated and collide withinert gas molecules to produce fragments. This techniqueis often used in mass spectrometers that have multiplemass analyzers, and the collisionally induced fragmentsare therefore called daughter ions since they are producedafter initial ionization and passage through the first-stagemass analyzer.

There are several types of mass analyzers, and someinstruments combine multiple mass analyzers. Time-of-flight mass spectrometers incorporate a simple design inwhich fragments are separated based on their velocities.Magnetic sector mass spectrometers separate fragmentsbased on the degree to which they are deflected in amagnetic field. Magnetic sector instruments are verysensitive, but cost and complexity have limited theirwidespread use in clinical laboratories. Instruments thatincorporate two magnetic sector mass analyzers (doublefocusing MS) can achieve very high resolution, and areuseful for making accurate mass measurements. Massmeasurements with accuracy to 0.0001 amu are usuallysufficient to determine the exact empirical formula of aparent ion or fragment.

The most popular mass analyzer is the quadrupole,which uses a combination of static and oscillating (radiofrequency) electromagnetic fields to separate the ionsproduced in the ion source. Quadrupole instrumentsare relatively inexpensive, have<1.0 amu resolution, andhave detection limits for most compounds in the picogramrange. Multiple quadrupole instruments have also beendesigned, their principal advantage being the ability toanalyze mixtures of compounds.

A variation on the quadrupole mass analyzer is theion trap mass spectrometer. The principal differencebetween a quadrupole analyzer and an ion trap isthat the former filters ions by creating an oscillatingelectromagnetic path through which the ions travel,whereas an ion trap confines the ions with the oscillatingelectromagnetic field. An advantage of the ion trapmass spectrometer is its sensitivity, since ions of aparticular mass can be accumulated, then released to thedetector – the ion yield is greater than that achievableby the quadrupole design. Ion trap instruments costabout the same as quadrupole instruments, and aremore sensitive, but also have two disadvantages: massspectra obtained on ion trap instruments do not always

correspond closely with reference spectra generated byquadrupole or magnetic sector instruments; and ion trapinstruments are, generally, less precise for quantitativeanalysis than are quadrupole instruments. Nevertheless,ion trap mass spectrometers are used in many of thesame applications as quadrupole instruments. Multiplemass analyzer instruments using ion traps have also beendesigned; usually the ion trap accumulates a particularion, and a quadrupole is used to subsequently measurethe daughter ions.

Most mass spectrometers use an electron multipliertube as the detector, although the design may be modifiedwith dynodes in order to measure both positive andnegative ions.

2.3 Combined Gas Chromatography and MassSpectrometry

The combination of GC and MS is one of the mostuseful and versatile analytical configurations availablefor measuring organic molecules. Although in principleany gas chromatograph and mass spectrometer could becombined, the most popular configuration nowadays is acapillary gas chromatograph with a split/splitless injectorand a quadrupole mass spectrometer using electronimpact ionization. This configuration is especially usefulfor measuring drugs in body fluids, when unequivocalidentification is necessary. Addition of isotope-labeledinternal standards (isotope dilution MS) is an accuratemethod for quantitative analysis as well.

GC/MS applications are limited to analytes withsufficient volatility to be vaporized in the GC injectorand oven; temperatures in these devices do not ordinarilyexceed 300 °C. A wide variety of clinically importantbiomolecules do not meet this criterion, including proteins(enzymes, hemoglobin, immunoglobulins, most peptidehormones), lipids, and some steroid compounds. Insome cases involving polar analytes, perfluorinated ortrimethylsilyl (TMS) reagents can be used to generatevolatile derivatives of these compounds, which can beseparated on a gas chromatograph.

3 SPECIMEN PREPARATION

GC and MS applications both have specific specimenrequirements; GC requires volatile specimens and MSrequires pure specimens. As a consequence, biologicalspecimens are rarely suited to analysis by GC or MSwithout some degree of preanalytical manipulation.Specimen preparation may involve extraction of theanalyte from a complex matrix, separation of componentsby a chromatographic procedure, generation of volatile


derivatives, or dissolution into a special matrix prior tousing a solid sampling technique.

3.1 Considerations for Biological Specimens

Most biological specimens are complex matrices withthousands of constituents, many of which may not beknown. Blood is the most commonly analyzed biologicalspecimen, and it contains both soluble and cellular com-ponents. Even within the noncellular fraction of blood(serum or plasma), there exist many highly lipophilic com-ponents suspended in micellar carriers. Water-solublecomponents of blood include small ions (e.g. sodium,potassium and chloride) as well as immunoglobulinswith molecular weights exceeding 500 kDa. Many con-stituents in blood are complexed with other components;for example, about half of the calcium in blood is boundto albumin. Many drugs are bound to protein carriers,and some ions, such as iron and copper, exist almostexclusively in a complexed form. Finally, the concen-tration of blood components can vary from millimolarquantities (glucose, electrolytes, phosphate) to pico andfemtomolar quantities (insulin, some toxins). Urine is alsocommonly analyzed in clinical laboratories, and containsmany waste products of metabolism (such as urea), excessdietary components (sodium, phosphate), and other pur-posefully excreted bodily constituents, such as drugs ornonuseful dietary components. The concentration of theconstituents in urine vary based on the rate at which theyare filtered and excreted by the kidney, as well as thetotal volume of urine that is produced, which is affectedby many factors. Hence, concentrations of normal com-ponents found in urine are generally more variable thanthe common constituents of blood, which are under ahigher degree of metabolic control.

An additional consideration, when interpreting theconcentration of constituents in biological matrices, is thatthe body is a heterogeneous matrix, so the concentrationof a particular constituent in the blood may not necessarilyreflect its concentration in other tissues. This is animportant consideration for many clinically relevantcompounds, such as therapeutic drugs, because they exerttheir principal effects at receptors in extravascular tissues.Hence, the ability to measure a constituent in the blooddoes not guarantee that the information will be clinicallyuseful. It is also important to consider the way a compoundis metabolized. This is especially true for drugs, manyof which are quickly modified in vivo by hydrolytic orglucuronidation reactions.

Other biological matrices include extravascular fluids(peritoneal, pericardial, synovial fluids), cerebrospinalfluid, saliva, feces, sweat, and in some cases keratinizedmatrices such as hair or fingernails. Although thesespecimens are less commonly used for clinical analyses,

each can be useful for a specific analytical purpose.Enzyme measurements in peritoneal fluid help distinguishbetween transudative and exudative processes, sweatchloride measurements are used to diagnose cysticfibrosis, abnormally high protein concentration in thecerebrospinal fluid is consistent with meningitis, andanalyses of hair and fingernails can reveal historicalexposure to drugs or certain heavy metals.

3.2 Preparation of Volatile Derivatives for GasChromatography

Organic compounds often contain carboxylic, hydroxyl,or amino functional groups that contribute to noncovalentintermolecular bonds, reducing the vapor pressure ofthese compounds. The low vapor pressure and, in someinstances, thermal lability of such compounds restricts theapplication of methods such as GC, which require vapor-phase analytes. One approach to adapting compoundswith low volatility to GC analysis is to synthesizederivatives that are more volatile. A diverse array ofreagents is available for generating volatile derivatives.The most popular of these reagents are polymethylatedsilyl and perfluorinated compounds.

In a typical strategy, a derivatizing reagent is chosento replace the hydrogen in a carboxylic acid or hydroxyfunctional group, generating the corresponding ester orether derivative. Polymethylated silyl moieties impartconsiderable nonpolar bulk to otherwise polar species,thereby limiting intermolecular associations and increas-ing volatility and thermal stability. Perfluorinated deriva-tives produce much the same effect, although the increasein molecular size is less dramatic. The choice of deriva-tizing reagent usually depends on the facility of thederivatization reaction and also the chemical propertiesof the resulting complex. Electron capture GC detectorsrespond most sensitively to halide-containing compounds,whereas high-molecular-weight fragments are most use-ful for mass spectral identification. Acidic perfluorinatedderivatizing reagents may react with the solid supportof the chromatographic column, accelerating the dete-rioration of column performance. In GC applications,high-molecular-weight silyl derivatives may lengthen theretention time unacceptably. Overall, the most appropri-ate derivative will strike the best compromise betweensensitivity and selectivity of the chromatographic method.When internal standards are involved, it is also importantthat the derivatization reaction does not selectively mod-ify the analyte at the expense of standard, or vice versa.

3.3 Solid Sampling Techniques for Mass Spectrometry

The sampling requirements for MS are more demanding,since specimen purity is essential. However, the ion source


of a mass spectrometer is operated under vacuum, socompounds with low volatility are more accessible than inpressurized systems like GC. Many biological compounds,however, are thermally labile, so techniques must bedevised to vaporize these analytes at low temperatures.Solid sampling methods are designed to produce vaporstate molecules without sacrificing the integrity of theanalyte, upon which mass spectral identification depends.

Most solid sampling techniques for MS rely on physical,rather than thermal, means to produce vapor statemolecules. Electrospray ionization uses a concentratedcoulombic charge to disrupt solvent/analyte droplets,leaving vapor-phase analyte molecules for mass analysis.Thermospray ionization is a similar technique, butevaporates the solvent. FAB methods rely on the kineticenergy of bombarding particles to eject analyte moleculesfrom the solid matrix, and MALDI uses a laser to vaporizethe analyte-embedded crystalline matrix.

4 APPLICATIONS TO DRUGMEASUREMENTS

Detection and measurement of therapeutic and illicitdrugs in blood and urine is an important component ofany clinical toxicology service. Although testing for illicitdrug use is a common practice associated with drug-freeworkplace initiatives, clinical drug testing is distinct fromforensic drug testing in several respects. Forensic drugtesting is a highly regulated practice, in which the ana-lytical specifications are set forth in guidelines publishedby the specific agency (federal, state, independent) thatlicenses or accredits the laboratory to perform work-place drug testing. Clinical toxicology services, however,adhere to standards established by agencies that licenseor accredit clinical laboratories. Whereas the regulationof forensic laboratories is intended to standardize practiceacross all such laboratories, clinical toxicology laborato-ries have more flexibility to configure their services tomeet the specific needs of the patient population theyserve. As an example, a truck driver who must submit toa drug test that is required by the Department of Trans-portation will have his urine tested in a federally certifiedlaboratory for five different drug classifications. Federalregulations require that any positive result be confirmedby GC/MS analysis, and further specify the quantitativeamount of each drug above which the test is to be reportedas positive. In contrast, a physician evaluating a patientin an emergency department may only be interested inwhether opiates or barbiturates are involved. In this clin-ical setting, confirmatory testing is usually unnecessarysince the test result is parallel with the clinical conditionof the patient. In this setting, the quantitative threshold

for a positive result should be established based on theneed for clinical sensitivity.

There are also situations in which toxicological testsordered for medical reasons are used in legal proceedings,and in these cases the distinction between clinical andforensic drug testing is less clear. It would be routine foran emergency physician to request a blood alcohol ona trauma victim, in order to evaluate the risk of usinganesthesia. However, if the trauma occurred as a result ofan automobile accident, then the results of blood alcoholmeasurements are pertinent to civil or criminal chargesof driving while intoxicated. Another common situationin which medically relevant drug tests can be used forlegal proceedings is testing neonates for intrauterine drugexposure.

The vast majority of clinical drug tests, for both illicitand therapeutic drugs, are performed by immunochemicalanalyses, which are economical and easily automated.Commercial immunoassay reagents are not available forall clinically relevant drugs, though, and GC or GC/MSare attractive choices of analytical methods for measuringdrugs when automated methods do not exist. GC andGC/MS methods have an additional advantage overimmunochemical screening methods, in that they can beconfigured to screen for multiple drugs; immunoassaystypically screen for a single drug or class of drug(benzodiazepines, amphetamines, etc.).

4.1 Drug Screening by Gas Chromatography

4.1.1 Alcohols

Gas chromatographic analysis requires volatile analytes,and ethanol is a common example of a highly volatiledrug found in biological specimens. Ethanol is the mostwidely used alcohol, but clinical situations arise in whichanalyses for methanol, isopropanol, or ethylene glycol arealso important.

Numerous methods have been published for measuringethanol in blood..2,3/ Typical features of a GC ethanolmethod are summarized in Table 2. Since alcohol is avolatile constituent in biological matrices, interferencefrom other matrix components is not a serious problem.GC methods for alcohol can ordinarily be used for wholeblood, serum or plasma, or urine. In many states, the legalstatutes that establish alcohol concentrations above whicha person is considered intoxicated (per se laws) specifywhole-blood alcohol. Because whole blood has a higherlipid content, its alcohol content is 10–15% less thanserum or plasma, from which cellular components havebeen removed. Urinary alcohol measurements should beused for screening purposes only, since the concentrationis rarely of clinical relevance.

In some GC methods for ethanol, the specimen isinjected directly into the chromatographic column. This


Table 2 Gas chromatographic measurement of ethanol

Specimen type Whole blood, serum or plasma, urine; wholeblood is preferable, since per se lawstypically refer to whole-blood alcohol (seetext)

Injection Can be injected directly, or headspacesampling

Column Either packed or fused-silica capillarycolumn; stationary phase may be polar(e.g. Carbowax) or nonpolar

Detector FID is most commonStandardization Internal or external standards may be used;

internal standard should be nonconsumedalcohol, such as 1-propanol (methanol orisopropanol would not be appropriateinternal standards)

is a simple procedure since it requires no sample prepara-tion, but nonvolatile constituents in the specimen may foulthe chromatographic column prematurely. The preferredmethod involves headspace analysis. Headspace analysisfor alcohol typically involves incubating a specimen (60 °Cis common) in a closed container until equilibrium isreached and sampling (with an airtight syringe) the vaporabove the liquid. The headspace vapor is introduced intothe chromatographic column, and analysis proceeds thesame as if liquid had been injected. Headspace analysis isparticularly useful for highly volatile solutes, since highervapor concentrations increase the sensitivity of the mea-surement. Among the toxicologically relevant alcohols,ethylene glycol is the only toxicant that is insufficientlyvolatile for headspace GC analysis.

Alcohols may be separated with a variety of station-ary phases, and with packed- or fused-silica capillaryGC columns. Polar stationary phases such as Carbowax(poly(ethylene glycol)) are often used to separate alco-hols, but relatively nonpolar phenylsilicone or dimethyl-siloxane stationary phases can also be used. Isothermalchromatographic analysis of ethanol is usually performedbetween 50 and 100 °C, or the oven temperature can beprogrammed to increase, possibly to expedite the ana-lytical cycle time. Helium is a common choice of carriergas. FID provides the best combination of sensitivity andeconomy for GC measurement of hydrocarbons.

Although external calibration of a GC alcohol methodis possible, and may even be preferable in certaincircumstances (e.g. heavy workload volume), the matrixin which standards are prepared should match, as closelyas possible, the specimen matrix. This requirement canbe difficult to meet in biological specimens such as blood,since the exact composition is not known, and it can varyfrom one specimen to another. A better method is theaddition of an internal standard (1-propanol is a commonchoice).

A GC method has been described for measuringethanol, methanol, acetaldehyde, and acetone in bloodand urine;.4/ the method also measures ethanol in fecalspecimens. Whole blood, urine, or serum was injecteddirectly, without any pretreatment, on to a polar station-ary phase in a packed column. Buildup of nonvolatilematerial in the chromatographic column was minimizedby using an 8-cm pre-column packed with glass beads andstoppered with dimethylchlorosilane treated glass wool.Nitrogen was used as the carrier gas, and a FID measuredthe compounds. The method was calibrated against exter-nal aqueous standards. Measurement of ethanol in fecesinvolved homogenization and centrifugation to recovera liquid supernatant, which was analyzed in the samemanner as above.

Ethylene glycol is widely available as the principalingredient in antifreeze, and can be ingested accidentallyby children or pets, or intentionally by adults for suici-dal or alcoholic substitution purposes. Ethylene glycolhas a variety of toxic effects in humans, most notablyrenal failure caused by precipitation of oxalate and hip-purate crystals from the urine..5/ The methods appliedto GC measurement of ethanol, methanol, and iso-propyl alcohol are not applicable to ethylene glycolbecause of its low volatility..6/ One published methodfor GC measurement of ethylene glycol.7/ in seruminvolved pretreating the specimen with acetonitrile toprecipitate proteins. An internal standard, 3-bromo-1-propanol was added to the specimen. Ethylene gly-col and its metabolite, glycolic acid, were derivatizedwith N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA)before analysis. Chromatographic separation was per-formed on a 5% phenylmethylsilicone coated fused silicacapillary column, and the oven temperature was increasedfrom 90 to 170 °C at a rate of 15 °C min�1. Split injec-tion (1 : 20 split ratio) was used, and ethylene glycol andglycolic acid were measured with a FID.

4.1.2 Multidrug Profiles

Another useful application of GC in a clinical toxicologyservice is for multidrug profiling..8/ When patients presentwith altered mental status, particularly in an emergencyroom setting, clinicians must include drug intoxicationin their differential diagnosis. Conventional automatedmethods for drug screening have a limited scope.Immunoassays are available for most abused drugs,but are not always sensitive for every drug within aparticular classification. Thin-layer chromatography issometimes used for drug screening, but it is strictlya qualitative method and considerable experience isrequired to interpret the results. High-performance liquidchromatography (HPLC) with ultraviolet detection hasalso been applied to multidrug screening, but its use isnot widespread.


The performance of flame ionization and nitro-gen–phosphorus detectors have been compared, usingreference standards, with respect to their suitabilityfor drug screening by GC..9/ For nitrogen-containingcompounds, the nitrogen–phosphorus detector increasessensitivity by one or two orders of magnitude, com-pared to the FID. Retention times of drugs separatedon packed columns versus fused-silica capillary columnshave also been evaluated (also with reference standards),and the results were determined to be comparable..10/

Choice of a detection system may depend primarily onavailability and cost; a nitrogen–phosphorus detectoris more expensive, but eliminates interferences fromnon-nitrogen-containing substances in the specimen.Fused-silica capillary columns offer important advan-tages, including convenience and higher resolution.

Numerous methods have been described for screeningblood or urine specimens for drugs by GC..11 – 13/ Typicalfeatures of these methods include extraction of drugsinto an organic solvent, separation on a moderately polarchromatographic column, and measurement with a FIDor nitrogen–phosphorus detector. Retention indices areused to identify the different drugs.

Extraction of the drugs prior to analysis is necessarydue to the many nonvolatile constituents of bloodor urine, which can quickly result in a buildup ofresidue in the small bore of a fused silica capillarycolumn. Extraction procedures for basic drugs are usuallydesigned to generate the free base (uncharged) formof the drug, which is soluble in a nonpolar organicsolvent. Adjusting the pH of the specimen to a pointabove the pKa of the drug is necessary; the converse istrue for extracting acidic drugs. Neutral drugs should beextracted in either procedure. Liquid–liquid extractionsteps can be performed as many times as necessary togenerate a sufficiently pure sample, but the extractioninefficiency will ultimately compromise the sensitivityof the method. Liquid–solid extractions are also useful,and will be discussed later in this article. An attractivefeature of extraction steps, in addition to separation ofanalyte from other matrix constituents, is that the solventcan be evaporated, thereby concentrating the analyteand improving sensitivity. Because analytical sensitivityis an important consideration in drug screening, splitlessinjections are preferred, but not essential.

Temperature programming is usually necessary inorder to elute analytes with a wide range of retentionindices within a reasonable period of time. One approachto improving the chromatographic characteristics of thedetectable drugs is to generate methylated derivatives,which reduce the overall polarity of individual analytes.A method for generating methylated derivatives in thechromatographic column has been described..12/ On-column derivatization involves mixing the specimen with

a derivatizing reagent – tetramethyl ammonium hydrideis a common choice – so that the heat of the injector willcause the derivatizing reaction to take place. This methodis particularly useful for acidic drugs, which have poorchromatographic characteristics when the carboxylic acidis left exposed.

4.1.3 Gas Chromatography as a Reference Method

GC is a sophisticated analytical technique that ordinarilyrequires some sample manipulation and a considerabledegree of user training and experience in order toproduce reliable results. Most clinical laboratories favorautomated methods, due to their higher throughput. Forthis reason, GC applications in clinical laboratories – evenclinical toxicology services – are not widespread, but canbe useful adjuncts when personnel and clinical needsare sufficient to justify the investment. Perhaps a moreappropriate use of GC in a clinical laboratory is toverify the accuracy of routine automated methods. Anexample taken from the literature.14/ involves comparisonof four automated methods for measuring lactate with gaschromatographic quantitation. Lactic acid was extractedfrom serum and the butyl derivative was synthesizedbefore GC analysis on a capillary column coated withmethylsilicone as the stationary phase. The lactate resultsobtained by GC analysis, which was regarded as thereference method, were compared to four commercialautomated lactate assays; regression slopes were between0.99 and 1.06, with standard errors of the estimatebetween 0.12 and 0.23.

4.2 Quantitative Applications of GasChromatography/Mass Spectrometry

The most common application of GC/MS in a clinicalsetting is for toxicological analyses, most notably theconfirmation and quantitation of drugs in specimensthat had previously had positive immunoassay screeningresults..15/ Confirmatory analysis by GC/MS is requiredin forensic drug testing services, and can also be anoptional enhancement to clinical toxicology services.The value of confirmatory analyses for drugs is notas great in a clinical setting, though. For example,when a positive test for benzodiazepines (a class ofsedative/hypnotic drugs) is obtained in the urine of ajob applicant, it is important to know which particulardrug is present, in the event that the applicant mayhave a physician’s prescription for one of these drugs.However, an emergency physician who is concernedwhether a patient’s depressed mental state might be dueto oversedation may not require information about whichspecific benzodiazepine is present, since it is not likelyto influence the treatment of the patient. Numerousapplications have been described for measuring drugs


Table 3 Selected GC/MS reference methods

Analyte GC/MS method features Clinical importance

Creatinine.18/ Isotope dilution; filtration to remove proteins;purification with HPLC; MBDSTFA derivative;quadrupole MS

Creatinine is a waste product of muscle catabolism thatis commonly measured to evaluate renal function

Cortisol.20/ Isotope dilution; extraction into dichloromethane;purification by column chromatography; HBFAderivative; quadrupole MS

Cortisol is a mineralocorticoid emanating from theadrenal gland; it is measured to evaluate adrenalfunction

Triglycerides.25/ Radiolabeled standard; extensive specimenpreparation including alkalinization,liquid– liquid extraction, solid-phase extraction;BSTFA derivative; magnetic sector MS;chemical ionization

Serum triglyceride concentrations have been correlatedwith risk for coronary artery disease

Cholesterol.23/ Internal standard; hydrolysis; extraction; BSTFAderivative; quadrupole MS

Sterol derivative found in all animals; associated withrisk for coronary artery disease

by GC/MS, but the vast majority of these are used forforensic drug testing. In this article, the use of GC/MS asa reference quantitative method will be emphasized, aswell as its application to esoteric analytes.

Table 3 summarizes the features of several recentlypublished methods using GC/MS as a reference tech-nique. Reference methods are not devised with expedi-ency or convenience as a goal, and some of these methodsare quite complex. Reference methods are intended toprovide a means to validate routine methods, not for useas routine methods. Therefore, accuracy is the principalgoal in the design of a reference method.

4.2.1 Creatinine

Creatinine is a byproduct of muscle catabolism, and itsconcentration in blood is roughly proportional to musclemass. Creatinine has no physiological function, and iscontinually excreted by the kidneys. Therefore, it isa convenient indicator of renal function, since loss ofkidney viability will result in increased concentrations ofcreatinine in serum. Measurement of urinary creatinineconcentration can also be useful in assessing renalfunction. In the late nineteenth century, Jaffe discoveredthat creatinine reacted with alkaline picric acid to forma bright yellow complex, and this reaction formed thebasis of an analytical method for measuring creatininethat is still used today. The Jaffe reaction is notoriouslynonspecific; picric acid reacts with a wide array of normaland abnormal constituents of urine and serum..16/ Hence,a reference creatinine method is especially helpful inassessing the accuracy of various methods based onthe Jaffe reaction. HPLC has been applied to referencemeasurements of creatinine..17/ In one method combiningHPLC with GC/MS,.18/ creatinine was isolated fromserum by deproteinization followed by HPLC, and thetert-butyldimethylsilyl derivative was synthesized beforeGC/MS analysis. The special purification procedure was

necessary to remove any creatine, which is the hydratedform of creatinine. An isotopically labeled internalstandard was used to calibrate the method.

4.2.2 Cortisol

Cortisol is a steroid hormone produced by the adrenalcortex that has mineralocorticoid and glucocorticoidactivity. The principal function of cortisol is the regulationof carbohydrate metabolism. Excess cortisol production,which can result from several causes, produces a setof clinical symptoms known collectively as Cushing’ssyndrome. Subnormal cortisol excretion is consistentwith adrenal hypofunction, a clinical condition calledAddison’s disease, but more sensitive indicators exist forthis condition. Measurement of cortisol in serum is animportant factor in evaluating patients with suspectedadrenal abnormalities..19/ A variety of immunochemicalmethods are available for cortisol measurement, andseveral of them were compared with quantitative cortisolresults obtained by GC/MS..20/ Cortisol was extracted intodichloromethane after alkalinization, and an isotopicallylabeled internal standard was added for quantitation.The extract was purified by liquid chromatography, andderivatized with heptafluorobutyric anhydride prior toGC/MS analysis. The comparison revealed significantdiscrepancies between various commercial immunoassaysfor cortisol, underscoring the importance of a reliablereference method for this analyte.

4.2.3 Cholesterol and Triglycerides

Coronary artery disease is the leading cause of death inmost industrialized societies, and considerable effort hasbeen directed toward identifying individuals susceptibleto coronary disease. The most widely used biochemicalindicators of risk for coronary artery disease are serumcholesterol and triglycerides. Since these compoundsare so frequently measured, and the results used to


approximate risk for disease, it is important that themethods used to quantify cholesterol and triglycerides beas accurate as possible. Reference methods are helpful instandardizing results across different analytical methodsand laboratories.

The consensus reference method for cholesterol isbased on a method described by Abell et al..21/ in1952, which involves alkaline hydrolysis of cholesterolesters, extraction into hexane, and reaction with sulfuricacid to produce a colored adduct. Reference standardscreated by the Centers for Disease Control are basedon this method..22/ However, several excellent GC/MSprocedures for cholesterol measurement have beendescribed, and it is likely that GC/MS will eventuallysupplant the manual Abell technique as the preferredreference method. In a recently described procedure,.23/

isotopically labeled cholesterol was added to a serumspecimen before hydrolysis with methanolic potassiumhydroxide at 70 °C. Free cholesterol was extractedinto hexane, and derivatized with N-trimethylsilyl-N-methyltrifluoroacetamide before GC/MS analysis. Theaccuracy of the method was verified using a standardreference material obtained from the National Institutefor Standards and Technology. Regression slopes forcorrelation of the GC/MS procedure with four automatedcholesterol methods were between 0.97 and 1.03.

Triglycerides present a more difficult analytical chal-lenge, since they comprise a heterogeneous populationof acylglycerol species. The Centers for Disease Con-trol recognize a reference method for triglycerides, basedon extraction, alkaline hydrolysis to generate glycerol,and oxidation of glycerol to formaldehyde; formaldehydereacts with chromotropic acid to form a colored adduct..24/

As with cholesterol, a GC/MS procedure may eventuallysupersede the wet chemical method. A recent exampleof a GC/MS method.25/ involves extraction of trigly-cerides into a chloroform–methanol solvent, followed bysolid-phase extraction. Specimens were hydrolyzed withethanolic potassium hydroxide at 70 °C for 2 h, and iso-topically labeled tripalmitin was used as the internalstandard for calibration and quantitation. N-Methyl-N-trimethylsilyltrifluoroacetamide was used to generatethe TMS derivative of glycerol before GC/MS analy-sis. The identity of the derivative was verified by negativeion/chemical ionization MS, which produced a protonatedmolecular ion. For triglyceride standards, the GC/MSmethod produced results that deviated less that 0.3%from expected values.

4.2.4 Therapeutic and Abused Drugs

GC/MS confirmation of positive screening results forabused drugs is commonplace, whereas immunochemicalmeasurements of therapeutic drugs are rarely confirmed.

There are, however, GC/MS applications for less com-monly abused drugs and therapeutic agents for whichimmunochemical methods are not available. Three exam-ples will be considered here: lysergic acid diethylamide(LSD), busulfan, and mannitol/sorbitol.

LSD is a semisynthetic hallucinogenic drug that isderived from ergot alkaloids. Detection and measurementof LSD is difficult because of the potency of the drug;a typical dose may be between 40 and 120 µg, and the par-ent compound is extensively metabolized..26,27/ A methodhas been described for GC/MS measurement of iso-LSD,.28/ which is a co-product in the hydrolysis reactionthat produces LSD from isomeric ergot precursors. Priorto analysis, iso-LSD was extracted into 1-chlorobutaneafter alkalization, and the iso-LSD was converted toLSD by a hydrolytic isomerization reaction using sodiumethoxide. The product was purified by solid-phase extrac-tion, then liquid–liquid extraction prior to derivatizationwith BSTFA. GC/MS analysis included separation on a5% phenylmethyl siloxane capillary column, and electronimpact ionization. Quantitation of iso-LSD was basedon a nonisotopic internal standard, lysergic acid methyl-propylamide, and the method was capable of detectingiso-LSD at a concentration of 50 ng per liter of urine.

Busulfan (1,4-butanediol dimethanesulfonate) is achemotherapeutic agent that is used for marrow ablationprior to bone marrow transplant, but in high doses it canbe toxic to the liver and central nervous system..29/ Theseadverse effects are relatively common, and frequentlyfatal..30/ In an effort to correlate plasma concentra-tions of busulfan with clinical outcomes, a GC/MSmethod was developed for measuring busulfan in theplasma of patients being prepared for bone marrowtransplant..31/ Pusulfan (1,5-pentanediol dimethanesul-fonate) was added to the plasma specimens as internalstandard before extracting the drugs into ethyl acetate.Busulfan and pusulfan were iodinated prior to GC/MSanalysis. The chromatographic separation was on a non-polar capillary column (methyl silicon), and electronimpact ionization was used for mass spectral analysis.Quantitative analysis of busulfan had a between-dayimprecision of approximately 8%, and the limit of detec-tion corresponded to 2 µg per liter of plasma.

Mannitol and sorbitol are often added to irrigationfluids used in endoscopic procedures to adjust theirosmolality..32,33/ Absorption of these compounds canlead to complications, so it may be important tomonitor mannitol and sorbitol concentrations in patientsundergoing certain endoscopic procedures..34/ A methodhas been described.35/ for GC/MS measurement ofmannitol and sorbitol using dulcitol as internal standard.Specimens were deproteinized with ethanol, and then-butyldiboronate derivatives were generated by reactionwith n-butyl boronic acid. Chromatographic separation


was sufficient to resolve mannitol and sorbitol peaksfrom several potentially interfering compounds, includingD-galactose, D-arabinose, D-ribose, and D-xylose. Themass spectrometer was set for electron impact ionization,and identification of mannitol and sorbitol derivativeswas based on the fragment m/z D 253.

4.2.5 Fatty Acids

Fatty acids are important components of metabolicreactions, and concentrations in biological specimens canbe affected by the presence of metabolic disorders ordietary deficiencies. The general structure of fatty acidsis R�COOH, where R is an alkyl chain. Because fattyacid structures are analogous, measurement of individualcompounds in this group by chemical analysis is difficultwithout first separating the different fatty acids. GC/MSis well suited to fatty acid analysis, because it involvesboth separation and specific detection based on the massof the analyte.

Oxidation of fatty acids occurs in the mitochondria,and the ultimate product of fatty acid catabolismis acetyl-coenzyme A (CoA), which enters the Krebscycle. Fatty acid b-oxidation is an important sourceof energy when dietary sources are unavailable orinsufficient to meet physiological demands. There areseveral genetic disorders that result in compromisedto nonexistent mitochondrial b-oxidative capacity, andthese disorders often go unnoticed at birth since fattyacid utilization is negligible except under circumstancesof dietary restriction or unusual energy expenditure..36/

Measurement of fatty acids in plasma or urine can helpreveal metabolic deficiencies in the oxidation of thesecompounds.

In one recently described method,.37/ C-6 throughC-18 fatty acids were measured by a GC/MS proce-dure in plasma obtained from patients with variousmitochondrial fatty acid b-oxidation defects. Hydroxy-lated fatty acid analogs (3-hydroxyhexanoic acid and3-hydroxyoctanoic acid) were used as internal stan-dards. Plasma specimens were acidified before extractingthe fatty acids into ethyl acetate. The reactive car-boxylic acid ends of the analytes, and free hydroxygroups of the internal standards, were derivatized withN-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide(MTBSTFA) and N-methylbis(trifluoroacetamide)(MBTFA) in pyridine, and the mass spectra were gene-rated with electron impact ionization. This method wasa modified version of a previously described procedureinvolving bis-trimethyl derivatives..38/

In another procedure,.39/ C-12 through C-22 fatty acidswere measured in plasma from patients with three typesof disorders: medium-chain acyl-CoA dehydrogenasedeficiency; very long-chain acyl-CoA dehydrogenase defi-ciency; and mild-type multiple acyl-CoA dehydrogenase

deficiency. Nonanoic and nonadecanoic acids were usedas internal standards. The fatty acids were methylated.40/

and extracted into hexane prior to GC/MS analysis on anonpolar column with electron impact ionization.

A method has also been described.41/ for GC/MSmeasurement of fatty acid ethyl esters in order to quantifythe activity fatty acid ethyl ester synthase, which isthought to be released in alcoholic liver and pancreaticdisease..42/ Although enzymes can be measured directly,most assays quantify enzyme activity by measuring therate at which substrate is consumed or product isgenerated. Enzymes are important biological markers,because their release into the blood can be a sensitiveindicator of tissue damage. Ethanol abuse is commonin western societies, but the mechanisms by which itcauses organ damage are not well understood. Fattyacid ethyl ester synthase activity was quantified bymeasuring its product by GC/MS. Ethanol and palmitic,stearic, oleic, and arachidonic acids were added to serumspecimens and incubated for 4 h at 37 °C. Followingincubation, the fatty acid ethyl esters were extracted intoa chloroform–methanol phase, and ethyl heptadecanoicacid was added as internal standard. Fatty acid esters werepurified by thin-layer chromatography before GC/MSanalysis.

4.2.6 Urinary Organic Acids

There are numerous enzymes involved in metabolic path-ways using amino acids to synthesize compounds ofbiological importance, and genetic deficiencies in theseenzymes result in the accumulation, and excretion, oflarge amounts of the precursor compounds. Collectively,many of these inherited disorders are known as organicacidurias, since the appearance of abnormal concentra-tions of organic acids in the urine is used to detect thedisease. GC/MS is an attractive technique for urinaryorganic acid profiling, owing to its resolution, specificity,and sensitivity. Several methods for measuring urinaryorganic acids have been described; a few of these aresummarized in Table 4.

Methylmalonic aciduria can result from inheretedmethylmalonyl-CoA mutase deficiency or defects incobalamin (vitamin B12) metabolism..43/ Vitamin B12 canbe measured in patients with suspected genetic defectsin cobalamin metabolism, but the test is not specific..44/

Measurement of methylmalonic acid in blood or urine isa more sensitive test for disorders associated with vita-min B12 metabolism. A method has been described formeasurement of methylmalonic acid in urine collectedand dried on filter paper..45/ Measured sections of filterpaper were rehydrated, and organic acids were parti-tioned into ethyl acetate after acidification of the aqueousfilter paper extract. TMS derivatives were synthesized


Table 4 GC/MS methods for detecting organic acidurias

Analyte Derivative Internal Referencestandard

Methyl-malonicacid

BSTFA/TMCS Isotopic McCannet al..45/

Orotic acid BSTFA/TMCS Isotopic McCannet al..50/

Profile Ethoxylamine HCl,BSTFA andperdeuteratedBSTFA

Externalcalibration

Shawet al..53/

Profile Hydroxylamine,BSTFA/TMCS

Externalcalibration

Duezet al..52/

TMCS, trimethylchlorosilane.

prior to GC/MS analysis on a 5% phenylmethylsiliconecapillary column and electron impact ionization. Quant-itative measurements of urinary constituents can bedifficult to interpret since their concentrations can varydepending on the total volume of urine produced.Because the amount of excreted creatinine is relativelyconstant, its concentration is sometimes used to compen-sate for variations in total urinary output. In this methodfor urinary methylmalonic acid, creatinine was measuredin the aqueous filter paper extract, and the acid concentra-tion was expressed relative to creatinine concentration.Trideuterated methylmalonic acid was added as internalstandard.

Inherited deficiencies in urea cycle enzymes usuallyresult in an accumulation of intermediary compounds.One of these intermediates, orotic acid, is increasedin several urea cycle disorders, the most commonbeing ornithine transcarbamylase deficiency..46/ Severalmethods for chromatographic or GC/MS measurementof orotic acid in urine have been described..47 – 49/

In a procedure involving collection of dried urinespecimens,.50/ soluble urine components were elutedfrom measured sections of filter paper by shaking with5 mL of water, and creatinine was measured in theeluate for reasons explained above. An isotopicallylabeled internal standard was added to the extract, andorganic acids were extracted into ethyl acetate afteracidification. TMS derivatives were synthesized usingBSTFA/TMCS, and the analytes were separated on a5% phenylmethylsilicone capillary column. Mass spectrawere obtained by electron impact ionization.

More general approaches to diagnosing inheritedmetabolic disorders involve methods similar to drugscreens, where the intent is to detect multiple con-stituents and reveal unusual concentrations when theyexist. HPLC has been applied to screening urine for inher-ited metabolic disorders,.51/ and more recently GC/MSanalysis of volatile derivatives of organic acids in urine

has been used for this purpose. The difficulties associatedwith designing a useful analytical procedure increase inproportion to the number of analytes it is intended to mea-sure, and the challenge of diagnosing inherited metabolicdisorders is considerable. A recent paper described a typ-ical approach to quantitative profiling of urinary organicacids..52/ After addition of nonphysiological internal stan-dards (2-ketocaproic and dl-tropic acid) urine specimenswere incubated with hydroxylamine for 30 min, thenacidified with HCl before extracting organic acids intoethyl acetate. The organic acids were further purifiedby two cycles involving addition of ammonia–ethanoland re-extraction. TMS derivatives were generated usingBSTFA/TMCS–pyridine before GC/MS analysis. Twelveorganic acids were quantitated by comparison of the ionintensity for characteristic fragments with the internalstandards. Coefficients of variation ranged from 2.8%to 20.8% (within-run), and recoveries from standardsolutions varied from 78% to 251%; most of the organicacids, however, had acceptable recoveries between 90%and 110%. A few of the organic acids exhibited a non-linear response, which may explain the highly variablerecovery data.

In another urinary organic acid profiling procedure,.53/

ethoxime derivatives were synthesized by addition ofethoxylamine HCl prior to acid extraction and deriva-tization with BSTFA/TMCS. A novel feature of thismethod was the use of perdeuterated BSTFA in orderto identify unknown constituents that appeared in thechromatogram based on the ion shift observed in thecharacteristic fragment. Using this procedure, the Krebscycle intermediates, citramalic, tartaric, and 3-oxyglutaricacids, were identified in urine from two patients diagnosedwith autism. Arabitol and arabinose were also identified.

4.2.7 Blood Lead

GC/MS is most useful for qualitative and quantitativeanalysis of organic molecules, but this versatile techniquecan be applied to nonorganic analytes as well. An exam-ple of clinical and toxicological interest is lead. Lead isa potent biological toxin, exposure to which can occurfrom many sources, such as paints, ceramics, plumbing,and environmental contamination. Lead exposure is par-ticularly damaging in childhood, since it may result incentral nervous system toxicity, developmental delays,and learning disabilities..54/ In adults, lead poisoning ismanifested primarily in hematological disorders, includ-ing anemia, and hypertenstion. Screening of school-agechildren for lead toxicity has been suggested in order toidentify children at risk for lead-related problems.

Lead inhibits the activity of amino levulinic acidhydratase, an enzyme involved in the synthesis of hemefrom porphyrin, and the accumulation of protoporphyrin


in red blood cells is an indication of lead exposure,although it is less sensitive than direct measurementsof lead in whole blood. The most common methods formeasuring lead in blood involve electrothermal atomicabsorption spectrometry, or electrochemical methods..55/

However, a GC/MS method has been described.56/ formeasuring lead in blood. A low-abundance isotopeof lead (204Pb; natural abundance 1.48%) was usedas internal standard, and the method was calibratedagainst pure and matrix-matched standards. Whole-bloodspecimens were digested with concentrated nitric acid,and the free lead ions were complexed with ammoniumpyrrolidine dithiocarbamate. The lead complexes wereextracted into toluene, and 4-fluorophenylmagnesiumbromide (Grignard reagent) was added to generate thePb(FC6H4)4 complex. The complex was measured bynegative ion/chemical ionization MS using methane asthe reagent gas. Typical within-run imprecision for themethod was 0.5%, and quantitative results were possibleat concentrations as low as 1 µg per liter of blood. Thegeneral approach, involving complexation, extraction,and GC/MS analysis with an isotopic internal standard isadaptable to other heavy metals..57/

5 CLINICAL APPLICATIONS OF MASSSPECTROMETRY

It has been previously noted in this article that massspectrometric analysis requires pure specimens, and thislimitation complicates its application to bioanalyses,where complex matrices are commonplace. There arestrategies for overcoming this limitation, however, andthese techniques have been applied successfully to detec-tion and quantitation of biomolecules in complicatedmatrices. One strategy involves the use of high-resolutioninlet systems; GC is but one example that was discussed inthe previous section. Another analytical strategy involvesthe use of tandem mass spectrometers, the first stage ofwhich selects a pertinent fragment and the second stagemeasures daughter ion fragments produced therefrom.Also, mass spectrometric methods designed to measurelarge molecules using soft, or low-energy, ionization tech-niques are less affected by impurities in the specimenmatrix since large fragments with unique masses are oftenused for identification and quantitation. The potential ofmass spectrometric methods for clinical applications havebeen stressed by several reviewers,.58/ and a few repre-sentative examples of this promising technology will bediscussed here.

5.1 Screening for Inborn Errors of Metabolism

Like the GC/MS applications reviewed in the previoussection, MS alone has been applied to screening for

inborn errors of metabolism..59/ These screening meth-ods ordinarily involve detection of accumulated metabolicintermediates that result from deficiencies in enzymesnecessary for biosynthesis of essential biochemicals. Oneversatile profiling technique utilizing MS with electro-spray ionization.60/ involved spotting blood specimenson a piece of filter paper for stable transport to thelaboratory. A disk was cut from the filter paper, whichwas immersed in a methanol solution containing iso-topically labeled standards for 13 organic acids. Aftercold incubation, the methanolic extract was dried andreconstituted in butanolic HCl and incubated at 65 °Cfor 20 min. The derivatized extract was washed withhexane and dried prior to preparation for dissolutionin the electrospray solvent, consisting of an acetoni-trile–water mixture (80 : 20). A triple-quadrupole massspectrometer was used to sequentially select character-istic masses in the first sector, to allow argon-collisionalfragmentation in the middle sector, and to allow daughterion analysis in the final sector. A computer algorithmwas developed to tabulate the mass spectral data, per-form quantitative calculations, and flag abnormal results.This method facilitated the detection of a wide array ofmetabolic disorders, including phenylketonuria (PKU),maple syrup urine disease, and methioninopathies. Asignificant advantage of quantitative techniques suchis this one is a notable increase in the specificity ofthe procedure – falsely positive results are substantiallyreduced.

Quantitative methods for urinary constituents areinherently problematic because urine volume varies withsuch factors as liquid intake and renal function. Thevariability in concentrations of urinary constituents canbe compensated, to some degree, by measuring the ratioof one constituent to another, which is less affectedby changes in total urinary output. This approach hasbeen used in mass spectrometric procedures designed todetect PKU. PKU is an inherited deficiency in aminoacid metabolism that can result in mental retardationunless dietary restrictions are implemented at a veryearly age. Because PKU is comparatively common amonginborn metabolic errors, although its incidence is still lessthan 0.01%, newborn urine screening for this diseasehas been widely adopted. As with any screening testfor a rare disease, though, a slight underperformance inspecificity results in a large number of falsely positiveresults, and low predictive value..61/ Falsely positiveresults of screening tests for heritable diseases canresult in unnecessary anxiety and expensive follow-uptesting. A recent report.62/ describes a mass spectrometricmethod for detecting PKU. The clinical sensitivity andspecificity of the screening test was improved by usingthe ratio of phenylalanine to tyrosine. Tandem MS(MS/MS) was used to quantify phenylalanine and tyrosine


following initial screening for hyperphenylalaninemia bya fluorometric method. The MS/MS procedure confirmedPKU in less than 2% of specimens classified as positiveby fluorometry. Other studies have produced similarresults..63/

MS/MS has been applied to the detection of maplesyrup urine disease, a rare disorder that traces its nameto the sweet odor of accumulated branched-chain aminoacids in the urine. The disease is caused by a deficiencyin branched-chain a-keto acid dehydrogenase activity,which is responsible for the oxidative decarboxylationof leucine, isoleucine, and valine. Measurement of bloodleucine can reveal maple syrup urine disease at an earlyage when dietary restrictions are helpful in preventing thesevere mental and physical complications that accompanythe disease. In the MS/MS procedure,.64/ butyl derivativesof amino acids extracted from filter paper blood spotswere prepared by reaction with acidic 1-butanol. Deuter-ated alanine, valine, leucine, methionine, phenylalanine,tyrosine, and glutamine, as well as [15N1,13C1]glycine,were added as internal standards. The derivatized speci-mens were reconstituted in a methanol–glycerol solutionprior to MS/MS analysis. In the tandem mass spectro-meter, a cesium gun was used to vaporize and ionizeanalyte molecules, and relevant positively charged frag-ments were selected in the first mass analyzer region.Selected fragments were exposed to argon in the secondquadrupole region, and collision-induced decompositionfragments were measured in the third mass analyzer. Pre-dictable neutral-fragment losses in the collision sectoryielded high-abundance ions that were used for quanti-tation. Concentrations of leucine, isoleucine, and valinewere expressed relative to phenylalanine concentration.Analytical recoveries of leucine and valine ranged from86% to 105%, and the method imprecision was less than11.5% (between-run).

Two inherited metabolic disorders can result in accu-mulation of methionine in blood: homocystinuria andisolated hypermethioninemia. Homocystinuria resultsfrom cystathionine b-synthase deficiency, and hyper-methioninemia is most often caused by a deficiency inmethionine adenosyltransferase enzyme. Both disorderscan result in neurological and skeletal abnormalities.Dietary restrictions, and sometimes pyridoxine supple-mentation, can improve the prognosis when affectedindividuals are identified at an early age..65/ An MS/MStechnique has been described.66/ for measuring methion-ine in filter paper blood spots, and the method can be usedto screen newborns for the associated genetic disorders.In this procedure, amino acids are eluted with a methanolsolvent containing deuterated internal standards. Themethod is substantially the same as the procedure forthe branched-chain amino acids leucine, isoleucine, andvaline, described above, including derivatization with

acidic 1-butanol and use of deuterated internal stan-dards. FAB (cesium ion gun) was used to vaporize andionize the analytes from a methanol/glycerol solvent. Thespecimens used to develop this method had been previ-ously screened by a bacterial inhibition assay (Guthrietest). The best precision and correlation with bacterialinhibition results was obtained when quantitative resultsfor methionine quantitation were expressed relative tothe sum of leucine and isoleucine concentrations.

Smith–Lemli–Opitz (SLO) syndrome is an autoso-mal recessive disorder that is characterized by mentalretardation, facial dismorphism, and microcephaly, aswell as cardiovascular and urogenital malformations..67/

It has been suggested that SLO syndrome results froma deficiency in sterol 7-reductase, which catalyzes theconversion of 7-dehydrocholesterol to cholesterol..68/ Asa result, high concentrations of 7-dehydrocholesterol arefound in SLO syndrome; the structure of this metabolicintermediate differs from cholesterol only by two hydro-gen atoms. A mass-spectrometric method has beendescribed for measuring 7-dehydrocholesterol,.69/ usinga time-of-flight MS instrument. Neutral sterols wereextracted from whole blood into hexane, and applieddirectly to the MS target platform. An argon beamvolatilized the analytes, which were separated in thetime-of-flight mass analyzer. Stigmasterol was addedas internal standard for quantitation. The method wasable to detect 7-dehydrocholesterol in 3 patients diag-nosed with SLO syndrome, but not in 10 normal controlsubjects.

5.2 Protein Measurement

Measurement of proteins involves special analytical chal-lenges, because the chemical composition of proteinsconsists of only 20 amino acids. Therefore, all pro-teins have inherently similar chemical compositions. Thephysical properties of proteins are largely determinedby secondary, tertiary, and quaternary structures, whichestablish the spatial orientation of the primary amino acidchain. Purification techniques that separate componentsbased on size have been used for protein measurement,but these methods are cumbersome and usually impre-cise. Certain proteins have unique properties that canbe exploited in an analytical method – certain dyes, forexample, react with anionic proteins. Most specific pro-teins are measured by immunochemical methods, butantibody specificity is not absolute, and the chemicalsimilarities between proteins exacerbate the problem ofcross-reactivity. For these reasons, reference methodsfor specific protein quantitation are not widely avail-able, and MS has been proposed as a method to fillthat void.


5.2.1 Measurement of Trypsin Cleavage Products

Since measurement of high-molecular-weight speciesrequires specially adapted MS instruments, most approa-ches to MS protein identification and quantitation involvemeasurement of smaller protein fragments produced bydigestion prior to analysis..70,71/ A general method hasbeen described.72/ for quantifying specific proteins byisotope-dilution MS, and a prototype application wasdesigned to measure apolipoprotein A-1 in a standardreference material. In this procedure, the protein isdigested with trypsin, which reproducibly cleaves twofragments containing 7 and 11 amino acid residues thatare unique to the apolipoprotein A-1 parent molecule.Analogous deuterated oligopeptides were synthesizedand used as internal standards. Trypsin hydrolysis wasallowed to proceed for 24 h at 37 °C, after which aceticacid was added to quench the reaction. The digestionproducts were purified by HPLC before measurementon a tandem magnetic sector MS, using FAB ionization(cesium ion gun). The fragments used for MS analysiswere characterized by peptide mapping and sequencingby HPLC and nitrogen analysis.

5.2.2 Plasma Renin Activity

Renin is produced in the juxtaglomerular cells inthe kidney, and its function is to catalyze the cleav-age of angiotensinogen to produce the decapeptideangiotensin 1. Angiotensin-converting enzyme cleavestwo amino acid residues from angiotensin 1 to produceangiotensin 2, which is the physiologically active vaso-pressive form. Angiotensin 2 is rarely measured, becauseits concentration is very low and it is rapidly degraded intoinactive oligopeptides. Therefore, renin activity is custom-arily assessed by measuring angiotensin 1 concentration.In hypertensive patients, plasma renin activity may helpidentify whether the cause is renovascular or adrenocorti-cal in origin. Immunoassays have been developed for mea-suring angiotensin 1,.73/ but antibodies may react non-specifically with other endogenous angiotensins. HPLChas also been used for angiotensin 1 measurement,.74/ butmost detection systems for HPLC lack the sensitivity tomeasure clinically relevant angiotensin 1 concentrations.

An electrospray ionization/mass spectrometry (EI/MS)method has been described for measuring plasma reninactivity by quantitating angiotensin 1..75/ This methodinvolves incubation of plasma specimens for 18 h at 37 °Cto allow the renin-catalyzed conversion of angiotensino-gen to angiotensin 1 to occur. A Leu! Val substitutedangiotensin 1 analog was used as internal standard; thevaline substitution increased the molecular weight by14 amu. Analytes were extracted from plasma on asolid-phase C18 column, and then purified by reverse-phase HPLC. MS analysis was performed on a tandem

mass analyzer with electrospray ionizer operating in thepositive-ion mode. The limit of detection for the proce-dure was 0.14 ng of angiotensin 1 per milliliter of bloodper hour, and coefficients of variation were less than 15%.

5.2.3 Hemoglobin

Hemoglobin is a tetrameric 64-kDa protein that isthe principal oxygen carrier in blood. The hemoglobintetramer is usually composed of two a-subunits, andtwo non-a-subunits, which may be pairs of b-, g-, d-,or e-chains. The combinations of paired subunits giverise to several forms of hemoglobin, most of which dis-appear before or shortly after birth. The predominantform (approximately 96%) of hemoglobin in adults isa2b2, and is designated HbA. Hemoglobins can be post-translationally modified by nonenzymatic covalent addi-tion of sugar molecules at the N-terminal valine residuesof the b-chains. The modified form of hemoglobin hasbeen called glycated hemoglobin, and when the sugar isglucose, the complex is designated HbA1c. HbA1c mea-surement is useful because its formation is regulatedby blood glucose concentration, and therefore abnor-mally high concentrations of HbA1c reflect persistenthyperglycemia..76/ A variety of methods exist for mea-surement of HbA1c, including affinity chromatography,immunoassay, electrophoresis, and HPLC. There is con-siderable variation, though, in quantitative HbA1c resultsobtained by these methods,.77/ possibly due to ambiguitiesin the definition of what HbA1c really is. Unlike chromato-graphic and immunochemical methods, MS is capable ofidentifying specific molecular species, and therefore itsapplication to HbA1c measurement and standardizationhas been suggested.

A proposed candidate reference method for HbA1c.78/

involves isolation of red blood cells by centrifugation,followed by lysis in hypotonic solution. Hemolysateswere mixed with buffer and cellular debris wasremoved by centrifugation. Hemoglobin was enzymat-ically cleaved by endoproteinase Glu-C, generatingan N-terminal hexapeptide that was unique to thehemoglobin molecule and contained the sugar residuefrom glycated hemoglobin species. Endoproteinase cleav-age products were separated by HPLC before massspectral measurement on a single-stage quadrupoleinstrument with electrospray ionization. The method wascalibrated with external standards purified by a combi-nation of cation exchange and affinity chromatographicprocedures. Results of the MS method were comparedto HPLC measurement of HbA1c, and the two methodsproduces nearly equivalent results.

A procedure similar to the method described abovehas been used to detect a genetic hemoglobin variantthat is more facile toward glycation..79/ The hemoglobin


Rambam variant, which is characterized by a Gly!Asp substitution at position 69 in the b-subunit, wasdetected by measuring the mass shift of 58 Da thatresulted from the substitution. The single amino acidsubstitution apparently promotes glycation of b-chainlysine residues, which alters the results of conventionalHbA1c measurements.

5.2.4 Albumin

Albumin is the predominant protein in blood, andserves many functions including regulation of plasmaosmotic pressure and as a carrier protein for a varietyof blood constituents. Numerous genetic variants ofalbumin have been reported, but interest in thesevariants is more academic than clinical since they donot ordinarily result in clinical disease. Nevertheless,precise measurement of albumin and its genetic variants isdifficult for the reasons described above: its compositionis comparable to other proteins, it is not significantlymodified post-translationally with unique compounds thatmight be easily detectable, and genetic variants differonly slightly, often by the substitution of a single aminoacid residue. Mass spectrometric analysis of albuminaffords the opportunity to resolve the many variantsof albumin.

EI/MS has been used to quantify albumin and some ofits variants..80 – 83/ In one procedure,.83/ albumin was puri-fied by dialysis and size exclusion gel chromatography ona DEAE–Sephadex column. Eluates were collected andconcentrated by filtration and redialyzed. Agarose gelelectrophoresis indicated greater than 95% purity of thealbumin specimens purified in this manner. Purified speci-mens were dissolved in the electrospray buffer, consistingof acetonitrile–formic acid (500 : 1) before analysis. Theprocedure was calibrated with standards prepared fromhuman a-globin and hemoglobin. Using this method,multiple forms of albumin were demonstrated in patientswith heterozygous albumin variants. The method wasable to resolve mass differences associated with the pointmutations 177Cys! Phe (44 Da), 1Asp! Val (20 Da),and Arg-albumin (160 Da), as well as more significantmutations involving carbohydrate and oligosaccharidemodifications.

5.3 Drug Measurements

5.3.1 Tacrolimus

MS using non-GC inlet systems has also been appliedto drug analyses. One reported example is the use ofliquid chromatography and electrospray ionization todetect an immunosuppressive drug, tacrolimus (FK506),in blood..84/ Immunosuppressive therapy has becomestandard practice in organ transplantation programs, and

the availability of potent immunosuppressive agents maybe largely responsible for the success these programshave achieved. Immunosuppressive drugs, however, arecharacterized by a narrow therapeutic index, and thepotential for toxicity from these drugs is correspondinglyhigh..85/ Therefore, frequent monitoring of blood levelsof immunosuppressive drugs is an important componentof this type of therapy..86/ Immunochemical methods fortacrolimus are problematic, because of cross-reactivityof the antibody with metabolites of the drug, manyof which may be inactive..87/ Therefore, quantitativeresults of immunoassays can give misleading resultswhen the concentrations of inactive metabolites are high.HPLC methods can overcome this limitation, but mayinvolve complicated and lengthy specimen preparationprocedures..88/ In an MS procedure, blood specimenswere mixed with an acetonitrile–water solution toprecipitate proteinaceous material. Drugs were purifiedwith a solid-phase extraction column, and reconstitutedin the liquid chromatograph mobile phase. Mass spectralanalysis was performed on a triple quadrupole instrumentwith an ion-spray interface to the liquid chromatograph.Collisional dissociation in the middle quadrupole wasinduced by argon gas. The method was calibrated with aseries of external standards.

5.3.2 Anabolic Steroids

Drug-use in athletics has received much attention, andsports drug-testing laboratories have responded withincreasingly sophisticated methods for detecting the pres-ence of performance-enhancing drugs. Anabolic steroidsare particularly difficult to detect, since many of theabused compounds are endogenous. Metabolic modifi-cation of steroid compounds also complicates efforts todetect illicit use. Finally, detection of many anabolicagents requires highly sensitive analytical methods..89/

Ion trap mass spectrometers have the ability to sequesterindividual fragments, and coupling an ion trap to aconventional quadrupole mass analyzer allows measure-ments that are both highly selective and very sensitive.The ion trap MS approach has been applied to work-place drug testing,.90/ and to measuring anabolic agentsin urine..91/ Common features of these applicationsinclude solid-phase extraction to isolate the drugs fromurine matrix and addition of b-glucuronidase enzyme tohydrolyze glucruonyl conjugates. The drugs were deriva-tized with BSTFA before injection in the ion trap massspectrometer.

5.3.3 b-Blockers

Along with anabolic steroids, another class of drugs thathas been banned by the International Olympic Commit-tee and International Sports Federation is b-blockers,


which have b-adrenoceptor antagonistic activity. A com-bined liquid chromatography/electrospray mass spectro-metric method has been described for measuring severalof the commonly used b-blocking drugs..92/ The drugswere extracted from serum or urine with a solid-phaseC18 column prior to liquid chromatography. The massspectrometer was interfaced to the liquid chromatographwith an electrospray ionizer, and selected ion monitoringwas used to measure fragments unique to the b-blockerdrugs and their metabolites.

5.4 Elemental Analytes

Because of the high temperatures required to volatilizemetallic elements, spectrometric methods for thesespecies typically involve flame or electrothermal atom-ization. Higher temperatures can be achieved usinginductively coupled plasma as the ion source, and MShas been used as a detector for this type of instrument.Urinary iodine,.93/ and titanium in spleen tissue.94/ havebeen measured using inductively coupled plasma massspectrometry (ICPMS).

Iodine is an essential element that is incorporated intothyroid hormones. Iodine deficiency is a well-describedclinical condition that is prevalent in many underdevel-oped areas of the world..95/ Monitoring urinary iodineis one method for detecting iodine deficiency, and anICPMS procedure for measuring urinary iodine hasbeen described..93/ Urine specimens were mixed withradioactive 129I as internal standard, and mathematicalcorrections were made for the conversion of 129I to sta-ble 127I. The accuracy of the method was verified withstandard reference materials. An advantage of ICPMSis that minimal sample preparation is necessary, sinceall organic material in the specimen is destroyed at the10 000 °C temperature of the plasma discharge.

Another application of ICPMS was developed todetect titanium in spleen tissue, which was sus-pected to emanate from a titanium-containing articularprosthesis..94/ Paraffin-embedded tissue was removed byheating, then rinsing with organic solvent. Delipidatedtissue was digested with hot nitric acid before ICPMSanalysis. Although results were mostly qualitative, theICPMS method confirmed the presence of titanium in tis-sues, correlating with abnormal histological findings thatraised the suspicion.

5.5 Characterization of Protein Epitopes

An epitope is the region of an antigen molecule thatis recognized by an antibody and is responsible for thenoncovalent interactions that result in the formation ofan antigen–antibody complex. Epitopes may comprisea continuous sequence of amino acids in the protein

molecule, or may include segments brought in close prox-imity of each other by the secondary or tertiary proteinstructure. Knowledge of these specific molecular regionsthat bond to antibodies is essential for understandingthe selectivity of antibody-based analytical methods, andfor developing effective synthetic vaccines. X-ray crys-tallographic data have been used to characterize theantigen–antibody complex and reveal complementaryregions, but this technique requires isolation of the com-plex in crystalline form; this is not always possible. Otherapproaches utilize chemical modification of candidateepitopes, structural variants, and synthetic peptides toprovide indirect information about the nature of theantibody–antigen complex. These methods are time-consuming and costly, and the results can be dif-ficult to interpret. An innovative method has beendescribed.96/ that uses MS to identify antigen frag-ments in the presence and absence of antibody byMALDI.

Alkylated human and hen lysozyme were prepared byincubation with 4-vinylpyridine, guanidine hydrochlor-ide, ethylene diamine tetraacetic acid (EDTA), anddithiothreitol in Tris buffer at 37 °C overnight. Thealkylated proteins were purified by HPLC, and thendigested with endoproteinase for 15 h at 37 °C. Themixture of proteolytic products was incubated with anti-lysozyme IgG for 2 h at 4 °C, and a portion of the resultingsolution was prepared for MALDI/MS analysis. A controldigest, without antibody, was processed in parallel. TheMALDI matrix consisted of a-cyano-4-hydroxycinnamicacid in acetonitrile and water. Mass spectral analysis wasperformed in the positive-ion mode with a time-of-flightmass analyzer.

The key to epitope mapping by this approach iscomparison of antibody-incubated antigen fragments withcontrol fragments in the absence of antibody. SinceMALDI is a soft ionization technique, antibody-boundfragments are less susceptible to vaporization. Antigenfragments that appear in control analyses but are absentin antibody-incubated preparations are presumed to bebound to antibody. Mass analysis provides molecular-weight resolution sufficient to identify the peptides boundto the antibody. In the reported experiment, a 13-residuefragment was identified as containing the epitope. Epitopemapping by MALDI/MS is a promising techniquethat offers the advantages of simplicity, economy andspeed.

5.6 Detection of Genetic Mutations

Although both proteins and DNA are polymericbiomolecules constructed from a few available subunits,analysis of DNA material is much more difficult becausethe phosphodiester-linked strands are less stable than


polypeptides and are prone to the formation of non-volatile salts. In addition, the amount of DNA availablefor analysis is usually very small, although amplificationtechniques can be used to generate sufficient materialfor analytical studies. DNA analysis has revolutionizedseveral areas of clinical laboratory science, includingmicrobiology, histology, and genetic screening. Mostmethods for DNA analysis involve hybridization withradioactive, fluorescent, or enzyme-labeled complemen-tary strands..97/ MS has been applied to identification andmeasurement of DNA probes, using low-energy ioniza-tion methods and mass determinations to unambiguouslyidentify relevant oligonucleotides..98,99/

Cystic fibrosis is an autosomal recessive genetic dis-order caused by a mutation in a gene that has beenmapped to chromosome 7. The gene, which codes fora protein called cystic fibrosis transmembrane regulator(CFTR), is over 250-kbp in length, and over 500 spe-cific mutations have been identified that cause disease.This high degree of allelic diversity complicates the taskof screening individuals for genetic evidence of disease,since the specific mutations exhibit different ethnic inci-dences. Whereas testing for one group of mutations mayidentify a high percentage of cystic fibrosis carriers inone population, its sensitivity in another population maybe quite low. Analytical methods capable of economi-cally screening for large numbers of mutations would bebeneficial. MS is a promising technique for this type ofanalysis, and methods have been described for detectingmutations associated with cystic fibrosis using MALDIand time-of-flight MS..100 – 102/

In one of these procedures,.102/ oligonucleotides com-plementary to several CFTR intron sequences weresynthesized and amplified by polymerase chain reaction;the amplification products were purified and used asdetection primers. Using a technique called primer oligobase extension (PROBE), primer oligonucleotides wereannealed to native DNA, and extension was promotedby polymerase enzyme so that the oligonucleotide waslengthened to include the mutated region. The result-ing oligonucleotides were measured by time-of-flight MS,which was able to identify nucleic acid substitutions basedon the mass of the fragment produced in the MALDI ion-izer. Advantages of this MALDI/MS method are thespecificity of oligonucleotide detection, and eliminationof the requirement to label probe fragments.

ABBREVIATIONS AND ACRONYMS

BSTFA N,O-Bis(trimethylsilyl)trifluoro-acetamide

CFTR Cystic Fibrosis TransmembraneRegulator

CoA Coenzyme AEDTA Ethylene Diamine Tetraacetic AcidEI/MS Electrospray Ionization/Mass

SpectrometryFAB Fast Atom BombardmentFID Flame Ionization DetectorGC Gas ChromatographyHPLC High-performance Liquid

ChromatographyICPMS Inductively Coupled Plasma Mass

SpectrometryLSD Lysergic Acid DiethylamideMALDI Matrix-assisted Laser Desorption/

IonizationMBTFA N-Methylbis(trifluoroacetamide)MS Mass SpectrometryMS/MS Tandem Mass SpectrometryMTBSTFA N-(tert-Butyldimethylsilyl)-

N-methyltrifluoroacetamidePKU PhenylketonuriaSLO Smith–Lemli–OpitzTMCS TrimethylchlorosilaneTMS Trimethylsilyl

RELATED ARTICLES

Biomolecules Analysis (Volume 1)High-performance Liquid Chromatography of BiologicalMacromolecules ž Mass Spectrometry in StructuralBiology

Clinical Chemistry (Volume 2)Clinical Chemistry: Introduction ž Atomic Spectrometryin Clinical Chemistry ž Drugs of Abuse, Analysis of žPharmacogenetic Testing ž Serum Proteins

Forensic Science (Volume 5)Mass Spectrometry for Forensic Applications

Nucleic Acids Structure and Mapping (Volume 6)Mass Spectrometry of Nucleic Acids

Peptides and Proteins (Volume 7)Capillary Electrophoresis/Mass Spectrometry in Peptideand Protein Analysis ž High-performance Liquid Chro-matography/Mass Spectrometry in Peptide and ProteinAnalysis ž Matrix-assisted Laser Desorption/IonizationMass Spectrometry in Peptide and Protein Analysis

Pharmaceuticals and Drugs (Volume 8)Gas and Liquid Chromatography, Column Selection for,in Drug Analysis


Gas Chromatography (Volume 12)Gas Chromatography: Introduction ž Column Tech-nology in Gas Chromatography ž Hyphenated GasChromatography

Mass Spectrometry (Volume 13)Mass Spectrometry: Overview and History ž ElectronIonization Mass Spectrometry ž Gas Chromatogra-phy/Mass Spectrometry ž Liquid Chromatography/MassSpectrometry ž Quadrupole Ion Trap Mass Spectrom-eter ž Tandem Mass Spectrometry: Fundamentals andInstrumentation ž Time-of-flight Mass Spectrometry

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