Tài liệu HPLC for Pharmaceutical Scientists 2007 (Part 7A) pptx

7.2 IONIZATION METHODS AND LC/MS INTERFACES Different physical principles can be used to separate and measure ionscharged particles with different mass-to-charge ratios under high vacuum

identifica-a lidentifica-arge volume of sidentifica-amples requiring ridentifica-apid identifica-and identifica-accuridentifica-ate identifica-anidentifica-alysis, with the speed

of analysis contributing directly to the drug discovery cycle time

As one of primer analytical techniques, mass spectrometry (MS) developedfrom nineteenth-century physics, starting with the pioneering work of J J.Thomson on the electrical discharges in evacuated tubes In 1913, Thomsonwrote “I feel sure that there are many problems in Chemistry which could betackled with far greater ease by this than any other method The method issurprisingly sensitive—more so than that of Spectrum Analysis—requiresinfinitesimal amount of material, and does not require this to be specially puri-fied .” Indeed, MS offers speed, high sensitivity and isotopic specificity Thistechnique separates mixtures of ions on the basis of mass-to-charge ratios,

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HPLC for Pharmaceutical Scientists, Edited by Yuri Kazakevich and Rosario LoBrutto Copyright © 2007 by John Wiley & Sons, Inc.

providing the molecular weight of a compound and its structural informationfrom fragment ions It is widely used for identification and quantification ofknown/unknown organic compounds Rapid development of MS in recentdecades has further expanded its role in the structural characterization ofsmall molecules in the drug discovery process [1].

In combination with chromatographic separation techniques, principally inthe form of high-performance liquid chromatography (HPLC) / MS (LC/MS),mass spectrometry has become the principal method of mixture analysis inpharmaceutical research and development [2] Early discovery research ofteninvolves library compounds analysis using high-throughput LC/MS methods.Identification and quantification of drug metabolites is essential in drugmetabolism and pharmacokinetic studies Structural characterization of impu-rities and decomposition products in bulk drug substances is an integral part

of pharmaceutical product development LC/MS combines the high-resolutionseparation capability of HPLC with MS detection and characterization ability,playing important roles in all these aspects of drug discovery process

7.2 IONIZATION METHODS AND LC/MS INTERFACES

Different physical principles can be used to separate and measure ions(charged particles) with different mass-to-charge ratios under high vacuumconditions, and this has resulted in a variety of mass spectrometers [3] In prin-ciple, the functioning of all mass spectrometers in generating mass spectrainvolves four steps: (1) introduction of the sample; (2) ionization of the samplemolecule to convert the neutral molecules to ions in the gas phase (ionizationmethod); (3) sorting of the resulting gas-phase ions by mass-to-charge ratios(mass analyzer); and (4) detection of separated ions Critical components of amass spectrometer include ion source and mass analyzer Depending ondesired applications and sample types, different mass spectrometers can be uti-lized to perform specific analytical tasks

7.2.1 Ionization Methods

An ideal ionization source for a mass spectrometer should provide a high ization efficiency and a high stability of ions for subsequent mass analysis bymass analyzers In addition, control of internal energy deposited on ions duringionization should be achievable in the ionization source to control the degree

ion-of fragmentation It is also desirable to couple ionization source with matographic separation techniques, especially with HPLC Various ionizationmethods have been developed over the years, including electron impact (EI),chemical ionization (CI), desorption ionization (DI), matrix-assisted laser des-orption/ionization (MALDI), desorption electrospray ionization (DESI), elec-trospray ionization (ESI), and atmospheric pressure chemical ionization(APCI) Note that ESI and APCI are part of LC/MS interfaces that will be

chro-discussed in separate sections Table 7-1 summarizes some characteristics of

different ionization methods

EI and CI are two early developed ionization methods They are extremely

useful for ionizing volatile compounds In EI process, molecules are ionized

by collisions with energetic electrons (typically 70 eV) produced from a heated

filament It produces highly reproducibly mass spectra with extensive

frag-mentation of molecular ions Thus, library searching with existing EI mass

spectra is possible for unknown identifications The nature of fragmentation

in EI often leads to lower abundances or absence of molecular ions On the

other hand, CI is a soft ionization method that generates mainly molecular

ions by ion/molecule reactions of regent ions with analyte molecules [4]

IONIZATION METHODS AND LC/MS INTERFACES 283

TABLE 7-1 Summary of Ionization Methods

Ionization

Method Ionization Agent Strengths Limitations

EI Electrons (∼70 eV) Extensive Limited to

fragmentation, volatile/

reproducible nonpolar spectra, searchable moleculeslarge reference

compound EIlibraries

CI Gaseous ions Abundant molecular Limited to

ions with nonpolar and controllable moderately fragmentation polar

molecules,limitedfragmentationAPCI Corona discharge/ Operative at Limited to low

gaseous ions atmospheric to moderately

pressure, easy polar interface to moleculesHPLC, abundant

molecular ions

DI Energetic particles Abundant molecular Difficult to

(atoms, ions), ions from high- interface to photons mass compounds HPLCESI Electrical/thermal/ Operative at Poor results for

energy pressure, easy molecules,

interface to HPLC, limitedmultiple-charged fragmentationions for large

biomolecules

Commonly used reagent gases include methane, ammonia, and isobutane Theinternal energy deposition or fragmentation of ions determines the appear-ance of mass spectra It can be controlled by selecting appropriate reagentgases In terms of proton transfer reactions in CI-MS, the relative proton affin-ity between the analyte and the reagent gas determines whether the analytewill be ionized The analyte with a higher proton affinity than that of thereagent gas will be ionized, while the analyte with a lower proton affinity thanthat of the reagent gas will not be ionized Furthermore, the difference inproton affinities of reagent gas and analyte is largely responsible for the extent

of fragmentation if ionization of the analyte occurs

EI and CI methods are complementary to each other, providing molecularweight and structural information As an illustration, Figure 7-1 shows an EI-

MS spectrum of mometasone furoate, an anti-inflammatory steroid drug A

very low abundant molecular ion at m/z 520 is visible However, the base peak

in the spectrum is the fragment ion at m/z 295 corresponding to the loss of

furoate ring, HCl, and a moiety of [COCH2Cl] Other fragment ions in thespectrum yield structurally characteristic fragmentations for this molecule Incontrast, a CI-MS spectrum of the same compound exhibits the protonated

molecular ion as the most abundant peak at m/z 521 along with some

frag-ment ions (Figure 7-2) The appearance of these two spectra clearly strates the utility of EI-MS and CI-MS methods

demon-An inherent limitation for EI and CI methods is the requirement that thesample analyzed must be volatile Both methods do not produce MS data for

Figure 7-1 EI-MS spectrum of mometasone furoate.

polar compounds One solution to this limitation is to employ DI methods toionize nonvolatile samples with high molecular weights [5] In the DI process,energetic particles or photons impact onto samples on a surface and result inthe liberation of intact molecular ions via selvedge region without direct trans-fer of the energy to the sample molecules The particle bombardment includeskeV atoms (e.g., Ar, fast atom bombardment [6]), keV ions (e.g., Cs+, liquidsecondary ion MS [7]), and MeV ions (e.g., plasma desorption [8]) Both fastatom bombardment (FAB) and liquid secondary ion MS (LSI) utilize largeexcess matrix for absorption, excitation, and relaxation of energetic particles,producing mainly molecular ions of interest They are well-suited for studies

of natural products and small polar compounds with molecular weights of afew thousand daltons (Da) Another important DI method is MALDI, whichemploys a UV or IR absorbing matrix in large excess with samples (5000 : 1)

to absorb the photon energy from laser irradiation [9] This method generatesmostly singly charged molecular ions with molecular weight as high as

500 kDa MALDI has a high ionization efficiency for large biomolecules withsupersensitivity in the range of low-femtomole level It has become one ofmost widely used ionization methods in biological mass spectrometry

The latest addition to DI methods is DESI [10] It directs an aqueous sprayfrom an electrospray apparatus onto the sample on a surface In the process,the fast nebulizing gas jet transports the charged droplets and impacts the

IONIZATION METHODS AND LC/MS INTERFACES 285

Figure 7-2 CI-MS spectrum of mometasone furoate using NH3as CI reagent gas

surface in the absence of matrix, carrying away analyte molecules Thisapproach has been successfully applied to analysis of small molecules and proteins The unique characteristic of DESI is that it operates under ambient conditions.All other DI methods as described above normally requirevacuum operation conditions, and sample manipulation during experiments isnot feasible DESI lifts the restriction on the vacuum constraints and can bevery flexible in carrying out novel experiments Potential applications includeforensic analysis, explosive detection, and biological imaging experiments intissues.

7.2.2 Historical View of Interfaces

A critical component of the LC/MS system is the interface that connects anHPLC system to a mass spectrometer The basic requirements for a success-ful interface include maintaining chromatographic performance (minimumadditional peak broadening), high transfer efficiency from LC to MS, and nodegradation in mass spectrometric performance Historically, a main challenge

in LC/MS interfaces was that high liquid flows from HPLC make it very ficult to maintain the high vacuum required for the function of a mass spec-trometer A number of different LC/MS interfaces have been developed overthe years to address this issue and overcome the difficulty [3]

to introduce liquids into a mass spectrometer was to minimize the amount ofliquid into an MS, removing solvent by the vacuum system and ionizing theanalyte in the gas phase The pioneering work carried out by Tal’roze et al.[11] described the simplest direct liquid introduction interface In their experiments, solvent was introduced into the mass spectrometer through acapillary at a flow rate below 1µL/min The ionization of analytes occurred by

EI The low flow rate used in the experiments was a limitation and would notgive good sensitivities for analytes In 1970s, McLafferty’s group employed

a direct liquid introduction (DLI) interface to directly introduce a small fraction (<1%) of the liquid from HPLC into the ion chamber of a CI massspectrometer [12] The solvent acted as the ionizing reagent The maintenance

of the vacuum was assisted by using large pump systems and differentialpumping Micro-and nanobore chromatography (<1-mm-i.d column) weresuitable for DLI A detection sensitivity of picogram level was achieved forfull-scan analysis

the moving belt system was based on the physical method of evaporation ofthe mobile phase through heat and vacuum that leave analytes as a thincoating on a continuously cycling polyimide belt The analytes were trans-ported from atmospheric pressure region to the vacuum of the ion sourcethrough differentially pumped vacuum locks Ionization methods used

included EI and CI for volatile analytes The system has excellent enrichmentsand efficiencies, although it is often limited to the analysis of compounds whichcould have been analyzed by gas chromatography (GC)/MS.

devel-oped by Blakley and Vestal [14] In their approach, a liquid flow from HPLCwas directed through a resistively heated capillary connecting to the MS ionsource The heat and vacuum would evaporate the solvent from a supersonicbeam of mobile phase produced in the spray, creating charged small micro-droplets These small liquid droplets were further vaporized in the heated ionsource Ions present in the ion source were then transferred to the mass analyzer, and residual vapors were pumped away

Ionization process in thermospray involves ion desolvation/evaporationfrom charged liquid droplets and gas-phase ion/molecule reactions Both pos-itively and negatively charged ions are formed in the process Volatile bufferssuch as ammonium acetate are often used as part of HPLC effluent Thesebuffer ions act as CI reagent ions to form either protonated or deprotonatedions The gas-phase proton affinity (for positive ion) or acidity (for negativeion) of the analyte relative to the buffers will determine whether the analytewill be ionized If the proton affinity of the analyte is lower than that of thereagent ion, the analyte will not be ionized This CI-like aspect of the ioniza-tion process results in thermospray mass spectra containing mostly molecularions When buffer is not used in thermospray experiments, an external ioniza-tion method is often applied, including EI filament and discharge ionization.These supplemental modes produce solvent-related CI mass spectra

A main advantage of thermospray is that it can handle commonly usedHPLC eluents at higher flow rates (up to 2 mL/min) and generate good resultsfor polar, nonvolatile, and thermolabile compounds However, the sensitivity

of the method is highly compound-dependent and not particularly attractive

to high-molecular-weight compounds

of FAB method [15, 16] In this modified method, the HPLC effluent withadded FAB matrix (usually 5% aqueous glycerol) is continuously transportedthrough a fused-silica capillary to the tip of a FAB probe residing inside of theion source The HPLC liquid with matrix deposited on the tip of the FABprobe is subjected to atom bombardment for ionization of analytes The matrixaddition can be done either pre-column or post-column, although post-columnaddition is preferred The acceptable liquid flow rate in continuous-flow FAB

is less than 10µL/min Flow splitting or the use of capillary chromatography

is often required in the experiments A major advantage in this method is thereduced chemical noise since much less matrix is used in continuous-flow FABthan in standard FAB experiments This has led to improved detection limits

to subpicomole range Significantly, this interface allows the LC/MS analysis

of biomolecules that are traditionally analyzed by DI methods

IONIZATION METHODS AND LC/MS INTERFACES 287

7.2.3 Common Interfaces

The early developed LC/MS interfaces as described above have played tant roles in the evolution of LC/MS interfaces However, their applicability,sensitivity, and robustness are very limited The overwhelming popularity ofLC/MS today is largely due to the development of atmospheric pressure ion-ization (API) interfaces, including ESI and APCI

1917 [17] He described how a high electrical potential applied to a capillarycaused the solvent to break into small droplets In late 1960s and early 1970s,Dole and co-workers attempted to generate gas phase ions from macromole-cules in solution using an atmospheric pressure electrostatic sprayer by ionmobility spectrometry [18, 19] In the late 1970s, Thomson and Iribarne suc-cessfully demonstrated the production of macro-ions from electrically chargeddroplets using MS [20, 21] The very first applications of ESI were reportedindependently by Yamashita and Fenn [22] and Aleksandrov et al [23] in the mid-1980s Now ESI has become one of the most successful ionizationmethods / interfaces used in mass spectrometry [24]

The basic ESI apparatus consists of a spray needle at high electrical tial (4–5 kV), a thermal/pneumatic desolvation chamber, and the vacuum inter-face (Figure 7-3) The ESI process is electrophoretic in nature It may involvethe generation of charged micro-droplets under a high electrical field and thesubsequent evaporation of droplets using either a drying gas (N2) or thermaldesolvation The first step of ion formation is the droplet formation at theneedle tip when the high electrical field causes ions of the same polarity to

poten-Figure 7-3 ESI source schematic diagram The ion formation is illustrated in the

pos-itive ion mode

form “Taylor cone” on the solution surface and emit charged droplets Thesecond step of ion formation is solvent removal, and its process is somewhatdebatable One theory is Dole/Fenn’s coulombic explosion When the initiallyformed droplets become smaller droplets due to evaporation of solvents, thesurface charge density increases and the coulombic forces exceed the surfacetension (Rayleigh stability limit), with the droplets breaking into smallerdroplets Further evaporation process with Rayleigh droplet fragmentationproduces analyte ions [22, 25].

One of the most important features in ESI is the formation of multiplycharged ions for proteins/peptides [26] Since a mass spectrometer measuresmass-to-charge ratios of a compound, the multiply charged ions will appear in

the mass spectrum at m/z values that are fractions of the mass (MW) of the

ion This allows the detection of high molecular weights of proteins/peptidesusing a standard quadrupole mass analyzer (3000-Da mass range) In addition,the detection of multiply charged ions provides precise measurements of molecular weights of proteins/peptides via the deconvolution method A massaccuracy of better than 0.01% can be achieved for proteins with masses up to

100 kDa [27] Another important characteristic of ESI is the softness of theionization It is a very mild process and can generate mainly molecular ionswith little fragmentation For small molecules, the singly charged molecularions usually dominate the mass spectrum The third characteristic of ESI is thesimplicity of the source design and its operation at atmospheric pressure,allowing ESI to be readily coupled to HPLC It is important to note that a lowflow rate (~200µL/min) of the sample solution is required in order to main-tain a stable spray in ESI Thus, flow splitters are often utilized in ESI-LC/MSapplications This does not reduce the concentration sensitivity of ESI sinceESI responses are directly related to the concentration of the analyte enter-ing the ion source However, the mass sensitivity can be substantially increasedwith a lower flow rate if the same concentration sensitivity is maintained

(c = m/v) This has led to the wide use of nano-spray (~ nL/min) LC/MS for

analysis of proteins and peptides, achieving femtomole sensitivity [24]

to ESI It was developed by Horning et al [28] in the early 1970s Figure 7-4illustrates a typical APCI source The sample solution is introduced into anozzle spray device similar to that used in ESI, but without the high electricalpotential applied to the nozzle The nebulizing gas (usually N2) is often added

to assist the desolvation/ionization process Although a heater at a ture of 400–500°C is used to vaporize solvents, minimal degradation of thesample occurs A corona discharge needle at a high voltage (3–5 kV) is respon-sible for producing a discharge current and inducing solvent ionizations Thegenerated solvent reagent ions react with analyte molecules via gas-phaseion/molecule reactions and produce analyte ions Clearly, the ion formationprocess is separated from solvent evaporation process in APCI (in contrast toESI), allowing the use of solvents unfavorable for ion formation For example,

tempera-IONIZATION METHODS AND LC/MS INTERFACES 289

low-polarity solvents generally used in normal-phase chromatography can beevaporated for APCI ionization Unlike ESI, APCI does not form multiple-charge ions for high mass compounds, and its response is more directly related

to the absolute amount of analytes APCI achieves optimal performance athigh flow rates (1–2 mL/min), making it ideal as the LC/MS interface for con-necting to conventional HPLC without flow splitters The other features ofAPCI include the appearance of CI-like mass spectra and suitability for analy-sis of volatile or semivolatile compounds

7.2.4 Special Interfaces

There are other LC/MS interfaces that are less commonly used than ESI andAPCI, but are often employed by researchers for analysis of nonpolar orneutral compounds, including particle beam and atmospheric pressure pho-toionization (APPI)

(monodisperse aerosol generation interface for chromatography), was oped by Browner and co-workers in the early 1980s [29] It uses a momentumseparator to eliminate volatile solvents and to transport analyte in the form

devel-of micro-aggregates particles to the EI/CI source devel-of a mass spectrometer ically, the LC effluent is forced into a small nebulizer using a helium gas flow

Typ-to form aerosol droplets These uniform droplets go through a desolvationchamber and evaporate into particles that are further separated from solventvapors by a multistage momentum separator The flow rate of liquid samples

in particle beam interface ranges from 0.1 mL/min to 0.5 mL/min The face is limited to relatively volatile compounds One of advantages in using aparticle beam interface is the database searching capability with the EI-MSlibrary for structural identifications

used in mass spectrometry to ionize a variety of compounds [3] The

forma-Figure 7-4 APCI source schematic diagram.

tion of ions involves the absorption of a photon by the molecule and ejection

of an electron from the molecule to form the radical cation The necessary dition for the ionization to occur is that the photon energy has to exceed theionization potential of the molecule of interest Like ESI and APCI, APPI usesnebulizer and vaporizer for desolvation The ionization occurs at atmosphericpressure with UV light source [30].The standard UV lamp has a photon energy

con-of about 10 eV that is sufficiently high to ionize most organic molecules.Common HPLC solvents and permanent gases usually have higher ionizationpotentials that will not be ionized This results in relatively noise-free massspectra, as opposed to ESI or APCI In some cases, analyte molecules mayexhibit higher ionization potentials (>10 eV) and the direct photoionizationmay not produce ions Then, addition of a large excess of a dopant such astoluene and acetone is necessary to yield charge carriers for ionizing analytes

of interest The undesired consequence of this dopant-assisted APPI is theincrease of background ions

The potential application of APPI includes analysis of compounds polar and neutral analytes) that are not effectively ionized by ESI or APCI.APPI appears to be less influenced by matrix suppression as seen in ESI orAPCI It can serve as a complementary ionization source to ESI/APCI

The basic function of a mass analyzer is to measure the mass-to-charge ratios

of ions (charged particles) and provide a means of separating the ions Theoperating principles of mass analyzers depend on interactions of charged par-ticles with electrical or magnetic fields Commonly used mass analyzers includemagnetic sector, quadrupole, ion trap, time-of-flight (TOF), and Fourier trans-form ion cyclotron resonance (FT-ICR) The combination of different massanalyzers can provide additional capabilities of performing mass spectrome-try/mass spectrometry (MS/MS) or tandem MS experiments for structuralcharacterization Table 7-2 lists some characteristics of various mass analyzers

7.3.1 Magnetic Sector

When an ion passes through a magnetic filed perpendicularly, it moves along

a circle with the radius where the centrifugal force is balanced by the netic force Essentially, the magnetic sector acts as a momentum-to-chargeanalyzer It brings a divergent beam of ions to focus (direction focusing) If allions have the same kinetic energy, the magnetic sector can behave as a massanalyzer The mass-to-charge ratios of ions are directly related to the magneticfield strength and the radius of the curvature, but inversely related to the accel-erating voltage in the ion source

mag-In a realistic situation, the ions produced in the ion source always have acertain distribution of ion kinetic energy that will impact the mass resolution

R(= m/∆m) In order to compensate for the distribution in ion kinetic energy,

an electrostatic analyzer capable of separating ions according to kineticenergy-to-charge ratios can be used in combination with magnetic sector Each

of these two devices independently focuses ions for direction, and togetherthey give velocity focusing—that is, equal and opposite dispersions for veloc-

ity This effect is termed double focusing (direction focusing and velocity

focus-ing), and the concept was realized in the early work of Aston [31] The doublefocusing instrument minimizes ion kinetic energy distribution and gives a highmass resolution independent of mass, providing accurate mass measurementcapability Other important features include large dynamic range and high-energy collision activation capability for structural elucidation studies.Although the magnetic sector instrument has played an important role in thestructural characterization of small molecules, its role in LC/MS is much lesssignificant because of sensitivity issue and the difficulty in interfacing with LC

7.3.2 Quadrupole

The quadrupole is a device in which electrical potentials of RF and DC areapplied to opposite pairs of a linear array of four parallel rods with hyperboliccross sections (Figure 7-5A) The ion motion under the electrical field can bedescribed by the Mathieu equation [32] In general, an ion moving through therod assembly only experiences the force in the plane normal to the direction

of ion motion (z-direction) Only ions that are stable in this plane will remain

in the rod assembly and eventually reach the ion detector For a given to-charge ratio of ions, the stability relies on the size of the rod assembly, oscil-lation frequency, RF voltage, and DC voltage The mass analysis is performed

mass-by sweeping DC and RF voltages, while maintaining their ratio and oscillation

TABLE 7-2 Characteristics of Mass Analyzers

Quantity Being Mass Range Mass DynamicMass Analyzer Measured (Da) Resolutiona rangeb

Magnetic sector Momentum per >104 >104 >106

chargeQuadrupole Path stability >103

Unit resolution >104

Ion trap Path stability >103 Unit resolution, >103

>103

at slowscan speedTime-of-flight Time >105 >104 >103

FT-ICR Frequency >105 >105 >104

a Mass resolution is defined as m/∆m, where ∆m is defined as mass difference at full width at

half-maximum (FWHM).

bDynamic range is defined as the range of either ion counts or sample concentration over which

a linear response is obtained.

frequency constant This mass-selective stability scan mode allows ions of ferent mass-to-charge ratios to be stable and pass through the device Thoseions of higher or lower mass than the desired ones are ejected from the rodassembly without passing through.The mass resolution of quadrupole depends

dif-on the ratio of DC-to-RF voltages Typically, a quadrupole is operated as amass analyzer with unit mass resolution

A quadrupole is small and relatively inexpensive It serves as an excellentcollision cell for collision activations of ions and ion/molecule reactions It canalso be used as a broadband ion transmission device A quadrupole is readilycoupled with other mass analyzers for MS/MS experiments One of the mostpopular configurations is a triple quadrupole mass spectrometer that hasfound wide applications in LC/MS and LC/MS/MS (see section on TandemMS)

7.3.3 Ion Trap

The quadrupole ion trap is a three-dimensional (3-D) analog of the linearquadrupole [33] It consists of two end-cap electrodes with hyperbolic crosssections and one ring electrode located between the end caps (Figure 7-5B).The RF voltage is applied to the ring electrode and the ground potential

is normally operated on the end caps A rotationally symmetric electric

Figure 7-5 Schematic diagram of (A) quadrupole and (B) 3-D quadrupole ion trap.

quadrupole field is generated in the device to trap ions with stable motionsdescribed by Mathieu equation The mass spectra are obtained by raising RFvoltage so as to cause the ions to become unstable and be ejected from thetrap through a hole in the end-cap electrode In this mass-selective instabilityscan mode, ions of increasing mass-to-charge ratios are ejected and detected

as the RF voltage is raised

The trapped ions possess characteristic oscillation frequencies The stablemotion of ions in the trap is assisted by the presence of a helium buffer gas (1 mtorr) to remove kinetic energies from ions by collisions When a supple-

mentary AC potential, corresponding to the frequency of a certain m/z ion, is

applied to the end-cap electrode, ions are resonantly ejected from the trap.This method of resonance ejection is used to effectively extend the mass-to-charge ratio of the ion trap Some other characteristic features of a 3-D iontrap include high sensitivity, high resolution with slow scan rate, and multiple-stage MS capability (see the section on tandem MS) In addition, it is inex-pensive and small in size As a result, a 3-D ion trap is widely used in LC/MSand LC/MS/MS applications

One of the inherent limitations to a 3-D ion trap is the ion storage ity because of the relative small volume inside the trap.The space-charge effectcan be significant when the ion population reaches above 106ions, impactingmass resolution, mass accuracy, sensitivity, and dynamic range In order toovercome this limitation, a 2-D linear ion trap has been designed to furtherimprove the performance of 3-D ion trap [34] Its quadrupole structure has ahyperbolic rod profile, similar to the conventional quadrupole rod In one ofthe designs in a 2-D linear ion trap, the quadrupole rod is cut into three axialsections (front section, center section, back section) Appropriate DC and RFpotentials are applied to the three sections to contain ions along the axis inthe central section of the device The ion detection is achieved by ejecting ionsout of the trap through a hole in the center section Dual ion detectors alongthe center section have been used to improve the sensitivity The advantages

of a 2-D linear ion trap over a 3-D ion trap are the increased ion storage ity (at least 10 times more than 3-D ion trap) and higher trapping efficiencies,leading to a better sensitivity and a larger dynamic range The use of 2-D linearion trap is gaining popularity in LC/MS and LC/MS/MS applications, either as

capac-a stcapac-and-capac-alone instrument or in combincapac-ation with other mcapac-ass capac-ancapac-alyzers (see thesection on tandem MS)

7.3.4 Time-of-Flight

In a time-of-flight (TOF) mass spectrometer [35], the ions generated in the ionsource are accelerated through a known potential and travel through a flighttube to reach the ion detector (Figure 7-6) The ion arrival time at the detec-

tor is measured, and it is directly related to the m/z values of ions It takes a

longer time for heavy ions to reach the detector, while light ions arrive at thedetector earlier