INSTRUMENTATION

       Dr. Lhost G.J.J.                                 

2.1  Magnetic Analyzers               2.4 Quadrupole Ion Trap 

2.2 Time of flight analyzers        2.5 FTICR 

2.3  Quadrupole Mass Filter      2.6  Mass Spectrometry/Mass Spectrometry    

 

2. Mass Analyzers

The primary function of all analyzers is to resolve an ion beam of mass m from another beam of m + Dm; this is a dispersive, or prism action.  The secondary function is to maximize the resolved ion intensities; this is a focusing, or lens, action. Upon their formation, all ions have slighly different kinetic energies due to the Boltzmann kinetic energy distribution and field inhomogeneities in the ion sources. These energy differences remain after the ions are accelerated out of the sources into the mass analyzers.  Velocity-focusing of some kind is needed to compensate for the ion energy inhomogeneity, e.g. by the electrostatic field in double-focusing magnetic analyzers or by delayed extraction in TOF analyzers.   The efficiency of a mass analyzer might be characterized by its degree of transmission and duty cycle.  In beam- type, scanning mass spectrometers (magnetic sector and quadrupole analyzers ) only a very small fraction of the ions issued from the ion source reaches the detector.  The degree of transmission refers to the fraction of ions that fall within the nominal m/z window of stable trajectories.  During mass scanning the conditions are set in such a manner that only ions having a m/z within a selected window can reach the detector, followed by a sequential changing of conditions so that all m/z values of interest will eventually be focused on the detector, one m/z value after the other.  The duty cycle is the ratio of the width of the transmitted m/z window to the total width of the m/z range of interest (meaning the fraction of all ions detected vs. all ions traversing the analyzer).  Hence, during scanning the duty cycle is usually < 1 %, i.e most of the ions are lost.  Where there is no scanning, the duty cycle is nearly 100 %, e.g in single ion monitoring.

2.1 MAGNETIC AND ELECTROMAGNETIC ANALYZERS     

      Consider an ion with mass m and charge q accelerated in the source by a potential difference Vs. At the source outlet, its kinetic energy is

 

2.1.1 Action of the Magnetic Field

If the magnetic field has a direction that is perpendicular to the velocity of the ion, the latter is submitted to a force FM as illustrated in figure 1 and its magnitude is given by

      FM =qvB

                 

         Fig. 1 Direction of the magnetic force upon a moving ion.

A circular trajectory with a radius r is followed by the ion so that the centrifugal force equilibrates the magnetic force

  For every value of B, the ions with the same charge and the same momentum (mv) have a circular trajectory with a characteristic r value. The ions are selected by the magnetic analyzer according to their momentum.

The kinetic energy of the ions at the source outlet is given by

                                                                      

      If the radius r is imposed by the presence of a flight tube with a fixed radius r, for a given value of B only the ions with the corresponding value of m/q go through the analyzer. Changing B as a function of time allows successive observations of ions with various values of m/q . If q = 1 for all the ions, the magnetic analyzer (which is fundamentally a momentum analyzer) can be used as a mass analyzer provided that the kinetic energy of the ions or at least their velocity is known.

Combining the two following equations

     The result is that ions with identical charge and mass are dispersed by a magnetic field according to their kinetic energy. In order to avoid this dispersion which alters the mass resolution, the kinetic energy dispersion must be controlled with an electrostatic analyzer.                                                                              

 

 

 

 

 

2.1.2 Action of the Electrostatic Field

Suppose a radial electrostatic field is produced by a cylindrical condenser. The trajectory is circular and the velocity is contantly perpendicular to the field. The centrifugal force equilibrates the electrostatic force according to the following equations, where E is the intensity of the electrical field.

                                                        

  The trajectory being independent of the mass, the electric field is not a mass analyzer but rather a kinetic energy analyzer, just as the magnetic is a momentum analyzer (mv = qBr). The electrical sector separates the ions according to their kinetic energy.

 

 

Six types of magnetic mass spectrometers and three double focusing instruments

Linked scans MS/MS

                    

 SELECTED IONIZATION MODES IN MASS SPECTROMETRY

Mode of ionizations for the JEOL Lcmate (Matsuda configuration)

A LC/MS system that offers classical high performance magnetic sector mass spectrometer technology in a benchtop package.   

FEATURES

 

3.  Mass spectra of immunosuppressive agents recorded with a

    JEOL LCmate instrument (Matsuda configuration)

 

3.1  Tacrolimus or FK506 (Data: Dr. Lhost G.J.J.)

             

 Tacrolimus (FK506) is a potent immunosuppressant having a 21-membered neutral macrolide structure (C44H69NO12). It was isolated from Streptomyces tsukubaensis , is commonly used to prevent rejection of organ transplant  and has been recognized to improve liver, kidney and lung graft survival rates relative to cyclosporin A (CsA).  This immunosuppressive drug possesses side effects that include nephrotoxicity , and neurotoxicity comparable to those seen with CsA.  Structural identification was determined chemically and by X-ray crystallography .  Tacrolimus undergoes extensive hepatic metabolism via cytochrome P450 isozymes of the P450 3A subfamily ..Metabolic studies of tacrolimus using rat and human liver microsomes indicate that O-demethylation and hydroxylation are metabolic pathways in liver microsomes   Also a tacrolimus C36-C37 dihydrodiol was isolated from induced rabbit liver microsomes  formed from an epoxide precursor under the influence of the hydrase enzymic activity and/or water.  A C19-C20 epoxide metabolite was isolated from pig liver microsomes proving that other dihydrodiols of tacrolimus do exist as well as other tacrolimus epoxides and most probably dihydrodiol epoxides.

      

   Electrospray ionization (ESI) mass spectra were obtained with a Jeol Lcmate benchtop LC/MS system (JEOL LTD, Tokyo, Japan).  The source voltage was set at 2.5 kV, the ESI/needle voltage at 2.5 kV, the needle current was 1.9 A, the desolvation plate temperature was 200C,  the orifice, ring lens and ion guide were set at 93 V, 207 V and 2.9 V, respectively and the flow rate of dry gas (N2) was 7 L/min.  The compounds (50 g) were dissolved in a mixture of acetonitrile -7.5 mM  ammonium acetate (50:50; sample concentration was 100 ng/L) and the solution was infused with the aid of a syringe pump at a flow-rate of 10 L/min. For linked scan mass spectrometry (B/E) helium was used in the collision cell at a pressure of 30 Pa corresponding to 80 % of the maximum pressure which may be applied. 

    

 

3.2  FK506 C36-C37 epoxide

 

Tacrolimus oxidation compounds were separated on a Macherey-Nagel nucleosil C8 column (5 m, length 250 mm, i.d. 10 mm) using a mixture of acetonitrile/water 50:50 as the mobile phase. The flow rate was adjusted at 2.5 mL/min, the oven temperature at 55 C and the UV detector was set at 208 nm.  

   

     

 

 

2.2. TIME- OF- FLIGHT ANALYZERS     

In time-of-flight (TOF) analyzers ions are separated according to their velocities.  Ions leaving the ion source are accelerated to 25 to 50 kV by a voltage applied to the final accelerating grid in the source and enter a 1 to 2m long, field-free drift tube D where they travel with various velocities toward an ion detector. The kinetic energy of all extracted ions is nearly constant, regardless of their masses.  Ions are formed in the source and accelerated to a constant kinetic energy zeV = mv2/2.  The time necessary for an ion to traverse the drift region (distance D) is t = D/v

The velocity of the drifting ions is inversely proportional to the square root of their m/z values.  Mass separation is achieved because the higher the mass, the lower the velocity of the individual ions, and conversely, the lower the ion's mass, the greater its velocity.  The flight time to the detector depends directly on the length of the flight tube and inversely on ion velocity.  The smallest ions arrive at the detector in a few s, followed by the larger ions at intervals a few fractional s later.  The m/z of each ion may be calculated from the time interval between the time it enters the fligft tube and the time it reaches the detector.     Because the ions must enter the drift tubes in discrete packages, MALDI sources, which produce ions in rapid , ns pulses, are ideal for TOF analyzers.   Ion beams emanating from sources that produce ions continuously, e.g., ESI, must be extracted in packets (pulses) preferably by injecting the ions orthoganally into the flight tube by " pusher" electrodes.  The range of m/z values leaving the source or arriving at the detector may be controlled with the aid of voltage pulses applied to ion-gates that are placed between the source and the flight tube or at the end of the flight tube prior to the detector.      Major advantages of TOF analysers are :

1. unlimited mass range, the practical upper mass limit is currently ~ 350 kDa.

2. The sensitivity of TOF analyzers is high because almost all ions produced are detected from each entering ion burst.

3. Possibility of high-speed analysis (e.g. complete mass spectra in 50 s.

Major improvements in resolution and sensitivity have resulted from the development of orthogonal injection, delayed extraction, use of reflectrons and hybridization of instruments.

Orthogonal Acceleration (Oa-TOF)

A strategy can be used which eliminates the initial kinetic energy distribution or, more precisely , the velocity distribution, along the TOF axis by orthogonal acceleration of the ions in a focused ion beam. 

a) Quadrupole injection   b)  ion storage area  c) two - stage orthogonal extraction and acceleration d) space focus plane    e) MCP  multichannel plate detector

A continuous ion beam emanating from an ion source is usually accelerated through an electric field resulting in a stream of ions having momenta proportional to the applied accelerating voltage and the m/z of the individual ions, but without temporal separation.  When the ion beam reaches the "orthogonal zone", a pulsed electric field gives the ions a second velocity component that is at right angle to the original velocity.  The pulsed ion beam then enters a TOF analyzer that provides temporal separation so that ions of differing m/z values arrive at the detector at different times. The pulsing rate may be as high as 30 kHz, which means that ~ 30000 spectra/sec can be collected and summed.  High performance data acquisition systems have been developed for combined CE/TOF systems which provide a spectral storage rate of 80 spectra/sec and a limit of detection (LOD) in the 10 to 25 amol range for continuous sample infusions.  Orthogonal acceleration permits the use of "continuous" ion sources, such as ESI with TOF.  The high sampling efficiency e.g. 30 % means a much better duty cycle than in scanning instruments, resulting in a substantial increase in sensitivity for obtaining full spectra.  TOF instruments with oa provide significant advantages in MS/MS operations.

Quadrupole injection (see figure QI-TOF)

The addition of an rf-only quadrupole for ion injection provides for more efficient collimation of the ion beam than an electrostatic lens as well as high ion transmission at the elevated pressures common for a ESI source.  In the collisional damping interface developed by Standing, collisions in the quadrupole region are used to reduce ion kinetic energies and ions are then injected into the storage region with a narrower energy bandwidth, which reduces the ion loss and mass discrimination that results from drift in the axial (nonorthogonal) direction.  The combination of quadrupole injection and orthogonal acceleration produce a TOF analyzer, which is relatively independent of initial conditions and, therefore, can accomodate almost any ion source. 

Delayed Extraction (DE)

Delayed extraction (time-lag focusing) is an in-source method of energy compensation.  Delayed extraction involves imposing a time delay between the ionization pulse and the application of the voltage to move the ions from the source into the flight tube .  First-order velocity focusing is achieved and resolution is improved because DE compensates for the initial velocity distribution of the MALDI generated ion packet.  This approach involves:

1. the expansion of the ions formed in MALDI ion sources into a field-free region between the repeller and the first extraction grid

2. a certain time lag or delay

3. a pulsed change in the relative voltages of the repeller and grid to create a potential difference and therefore extract the ions into the flight tube. 

Resolving power to 2500 may be achieved with DE alone.  The narrower peaks also improve sensitivity on account of improved signal-to-noise ratio.  DE loses effectiveness at masses > 20 kDa.

Reflectrons (Ion Mirrors)

While delayed extraction is an in-source method of energy compensation, the ion mirror is a postsource  compensation method for energy inhomogeneity.   The resolution of TOF analyzers is limited for several reasons including :

1. as the mass of the analyte increases , the difference in arrival times of ions at the detector becomes smaller and smaller, and more difficult to differentiate.

2. there is a spread in the temporal, spatial and kinetic energy distribution of the groups of ions of the same mass as they are are pulsed out of the source into the flight tube. This means that two ions of the same mass formed at different times, or at different locations in the source, will arrive at the detector at different times.

The reflectron is a homogeneous electrostatic ion mirror consisting of electrodes typically located in front of the detector used if the instrument is in the linear mode operation.  The polarity of the voltages on the electrodes is the same as that of the ions.  When the electrode voltages are adjusted to potentials slightly higher than that used to accelerate ions out of the ion source, ions arriving at the end of the drift tube experience a retarding potential , eventually come to stop and then accelerate in the opposite direction.  The reflected ions are usually made to travel a second length of drift tube set at a small angle to the first one ( V-shape),to be collected at a second detector. The ions fragmenting between the source and the reflectron are called post source decay (PSD) ions The V-shaped configuration is examplified by the

 JEOL LC-TOF MS or AccuTOF

_______________________

In coaxial reflectrons, the reflected ions move backward on the same axis and are detected with an annular plate detector close to the detector.Reflectrons achieve energy focusing of the ions as follows: when two ions of the same mass but with different initial energies (arising from the ionization process and the initial kinetic energy distribution) arrive at the reflecting field, the ions with higher energy penetrate the field to a greater extent, spend more time inside the reflectron, and exit later than the ions with lower kinetic energy.  Thus, the lower energy ions are refocused with those of higher energy so all ions of the same m/z reach a common plane at the final detector and are detected at the same time.  The result is reduced peak broadening and therefore higher resolution.  Another advantage of reflectrons is that they provide increased spatial separation of ions with different m/z values because their flight times are effectively doubled.  resolving power may be as high as 20,000, a 20 fold increase from the linear mode of operation.  However, the price of excellent resolution is reduced mass range and sensitivity.  The latter is due to loss of ions by collisions and dispersion in both reflectron and the second drift tube.  Accordingly, despite their improved resolution, reflectrons are often used for analytes of very high mass, particularly when they are present in trace quantities. 

 

 

Hybridation of instruments with the TOF mass spectrometer

It is not difficult to envision the quadrupole as the first mass analyzer in a tandem (hybrid) mass spectrometer, which uses an orthogonal acceleration TOF mass analyzer to record the product ions.  This is the intent of the QTOF mass spectrometer developed by Morris and commercially available from Micromass).

        Issues of conventional MALDI-TOF/TOF

           

       Single TOF mode

            - Low mass resolution

            - Larger mass dependencies in resolution and accuracy due to delayed extraction technique (High mass

              resolving power can only be  obtained in a limited mass range.

         

 

 

 

TOF/TOF mode

     - Low precursor ion selectivity 

                       1.  Interference with fragment ions from precursor ions nearby.

                       2. Unobservable minor fragment channels due to isotopic peaks

       -   PSD (Post Source Decay) ions are mainly observed.

      -   CID spectrum of compounds which are decomposed with difficulty by PSD can not be measured by

          the TOF/TOF system of ABI and Bruke,r  because their MS/MS system doesn't have enough energy to induce CID reaction.                   

 

The New JEOL SpiralTOF mass spectrometer (JMS-S3000)

The new SpiralTOF mass spectrometer has been introduced recently at the international mass spectrometry congress (IMSC 2009, Bremen).  The basic unit is the spiralTOF but additionally different options are possible corresponding to the following configurations basically illustrated herunder :

1.  SpiralTOF + TOF/TOF (MS/MS)                                                                 

2. SpiralTOF + Linear

3. SpiralTOF + TOF/TOF + Linear

Some technical characteristics of the instrument are summarized herunder:

Ion Source and Extraction

- MALDI

-Laser: Explorer (Spectra Physics)

- Delayed extraction

1st TOF:  Spiral TOFMS

- Flight Path :  17m (= 2.093 m x 8 turns)

- Ion Gate at 7th turn

- Detector: DM291 electron multiplier (ETP)

Collision Cell

-Differentially pumped

2nd TOF: Reflectron TOFMS

-1st deflecting system after Spiral TOFMS

-Reacceleration region including 2nd deflecting system

-OPIM: Flight Path 1.6 m

-Detector :  148821 (ETP)

 

Table of performance

TOF             Resolving power      50,000

                         Accuracy (IS)                 2 ppm

                         Accuracy(ES)             10 ppm

                         Sensitivity                    0.5 fmol

TOF/TOF     Precursor ion              2500                   

                         selectivity

                         Resolving power         2,000                                  Examples of Resolution in SpiralTOF mode :       

                         Accuracy                     0.1 Da                                  Examples of high precursor ion selectivity        :  

                        Sensitivity                    5 fmol                                  (HE-CID in TOF/TOF                                                                                                                                     

                        Fragmentation            HE-CID (20 kV)

 

 

 

2.3   Quadrupole Mass Filter     

A quadrupole field generated by four hyperbollic rods is interacting with an ion ejected from the ion source.  The rods are submitted to a direct current potential   U and  to a RF potential  (radio frequency potential)  Vcoswt  so that the potential F applied to the rods is  U +- Vcoswt .   By the action of this field , ions which enter the quadrupole mass filter oscillate in both x and y directions.                   

Parameters that affect the ion z motion are the U, V, w and radius r0 of the mass filter

A stability duagram of the quadrupole mass filter may be defined by two entities a and q taking into account the parameters  U, V, w and radius r0 of the mass filter.  

                                                     

 It is possible to define a and q values coresponding to stable trajectories inside the quadrupole mass filter.  Outside the stabikity diagram, the values of a and q are such that the ions will decharge on the rods.

If U, V and w are kept constant, a straight line may be drawn  in the stability diagram with a slope dependent on the ratio a/q = 2U/V         

 

                                           and             

Durind scanning of the instrument if the U/V ratio is maintained constant, all the ions will be selectively detected

  

 

                    Stability Diagram      

 

 

 

 

2.3.1   Tandem MS with triple quadrupole mass spectrometers

For accurate quantification , triple quadrupole mass spectrometers ,QqQ , have become a standard analytical tool foe LC-MS/MS applications.  In the MS/MS mode , Q1 is used as MS1 , q2 a RF-only device acts as a collision cell for CID experiments and Q3 is used to analyze the fragments.exting from q2 .  Selected ions emerging from Q1 are accelarated by an offset of ten electronvolts into q2 where collision with N2 or Ar gas at a pressure between 01-03 Pa occurs and the fragment ions are then anallyzed by Q3 .  Scan modes of triple quadrupole instruments are summarized herunder:

   Scan Mode   Operation of Q1  Operation of q2     Operation of Q3
product ion,

define m1

no scan, select m1  metastable or CID  scan up to m1 to collect the fragments
 precursor ion,

 define m2

 scan from m2 upwards to cover the precursors  metastable or CID  no scan, select m2,
 Constant neutral loss

define Dm

Scan desired range  metastable or CID scan range shifted by Dm to low mass

  

 

 

 

 

 

 

 

 

                                                   Triple quadrupole mass spectrometer

 

2.4    Quadrupole Ion Trap  (QUIT)             Trapped ions  

An  ion trap as illustrated is made of a ring electrode and two end - cap electrodes.   The two end - cap electrodes are electrically connected and when a   U + Vcoswt  potential is applied between the end-caps and the ring electrode a quadrupolar field is generated and inside the trap the ions are then following trajectories confined in a well defined space region. 

                                                                                                                  A stability diagram may also be defined by two parameters  a and q

                                                                                                                                                                                                   

                      

                                                       

     

 

 

 

 

 

Quadrupole ion trap with the two end-caps and one ring electrode

 

 

                                                                                                                                                      Stability diagram of a quadrupole ion trap

The potential applied to the ring electrode is generally a RF voltage, the Vcoswt component.and in this case the stabikity diagram related to the a and q parameters becomes a stability line related to the q values .

                                                                                           

For constant values of V, r0 and w, ions of different m/z values presents corresponding q values .  Ions may remain on the q axis at q values inversely proportional to their m/z values.  All the ions remain trapped in the trap region for appropriate values of m/z, V, r0 and w.  By scanning the

voltage V, the q values of different ions increases and when their q value reaches the limit of the stability diagram , ejection from the trap and detection occurs.

        

 

 

 

 

 

 

 

                  QUIT with external ion source and detector

                    ( illustration in the Z direction)

 

When a three-dimensional quadrupole electric field is created by applying superimposed rf ( ~106 Hz frequency) and d.c potentials (small voltages) on the ring electrode , the end caps being grounded, all ions are constrained into stable trajectories near the center of the trap in a predominant sinusoidal motion.  With the appropriate selection of the frequencies, all injected ions of different m/z values may be trapped with stable trajectories.  To measure an ion with a particular m/z value, the rf and/or the d.c component of the field is changed so that the ion trajectory along the z axis becomes unstable resulting in the ejection of the specific m/z from the trap volume, through holes in one of the end caps onto a detector.  A mass spectrum of an analyte including a scan of a selected mass range, is obtained by the sequential chjanging of frequencies, ejecting one m/z at a time.

2.4.1  Some specific properties of the ion trap (MS/MS)

Potentials can be chosen such that only ions with a selected mass m/z have a stable trajectory and remain trapped, while all others with different m/z values assume unstable trajectories and are ejected from the trap.The remaining trapped ions of the selectyed m/z may be exposed to multiple collisions with an injected buffer gas to produce fragmentations.and one of the major advantages of ion traps is the possibility to perform CID experments up to MSn.    Ion traps exhibit interesting behaviour for tandem-in-time experiments since they perform the steps of precursor  ion selection,, ion activation and acquisition of frament ion spectra in the same physical space.  In contrast to tandem-in-space instruments, tandem-in-time instruments do not support constant neutral loss and precursor ion scans.  In the ion trap, the ions are generated inside the trap or produced outside  and injected subsequently in the trap. 

2.4.1.1  Resonant ejection

 A suitable time is choosen in order to optimize the number of trapped ions avoiding a too great density of ions leading to space-charge effects.  The ion selection phase may be achieved by resonant ejection to remove ions of succesively increasing m/z value from the storage volume to produce a scan.  In the ideal situation, the motion of the ions in axial and radial directions are independent and their oscillations can be described by a radial and an axial secular frequency each of them being a function of the Mathieu trapping parameters a and q.  If a supplementary RF voltage which matches the axial secular frequency is applied to the end caps, resonant ejection of ions occurs.  Excitation occurs when the frequency of a supplementary RF signal matches the secular frequency of a trapped ion in the z direction.  Scans based on resonant ejection may be carried out from low to high mass or from high to low mass  allowing for the selective storage of ions of a certain m/z value by elimination of ions below and above that m/z value from the trap.   The collision of the preselected ion is again performed by resonance with the supplementary RF field with a frequency corresponding to that of ion motion but with an intensity maintaining the ion trajectory inside the trap walls.  The ion collides with the He atoms of the buffer gas present in the trap.   When sufficient internal energy has been acquiored, the ion decomposes to product ions which are ejected from the trap by the main RF scan and finally detected.  When the internal energy necessary to activate the decomposition at the lowest critical energy is reached, the ion fragments.  The sequence can be repeated by selection among the collisionally generated product ions of an ionic species of interest which is submitted again to collisionally induced dissociation and finally detected (MS3 ). and the process can be repeated more times (MSn)  In this way it is possible to obtain  a decomposition pattern at low energy with the production of fragment ions of high diagnostic value from the structural point of view.

2.4.1.2  Mass-Selective Stabikity Mode

All the ions are generated or admitted to the QIT but ions of one m/z value are trapped at a time when parameters of the QUIT are set at appropriate values.  By applying a negative voltage to one of the endcaps, the stored ions are pulsed out of the storage volume and reach the detector.  By addition of hundreds of single steps , one for each nominal m/z value, a full scan  mass spectrum is obtained.  This is the mass-selective stability mode which is no more used because of a lot of drawbacks ( poor sensitivity, slow speed)

2.4.1.3  Mass Selective Instabikity Mode

When all the ions are trapped within the QIT with the end caps grounded an RF voltage scan (V) is applied to the ring electrode causing consecutive ejection of ions in the order of their m/z values.  This is referred as the mass-selective instability ejection mode.

A timing sequence sequence is illustrated herunder.      

The trap is filled with ions from the electrospray source by dropping the repelling voltage on the gate lens to pass the ion beam.  Ions are trapped in the RF field using a low quadrupolar amplitude determined by the low mass cut-off.  After the accumulation time, the gate lens voltage is raised to stop ions entering the trap and the stored ions are "cooled" by collisions with the He bath gas to ensure that the ion cloud is positioned in a small packet at the center of the trap.  During the scan the quadrupolar amplitude and the dipolar amplitude on the endcaps are increased simultaneously to progressively eject ions of increasing m/z value through the exit endcap.  At the end of the scan the quadrupolar amplitude is dropped to zero, and no trapping occurs so trhat any ions remaining in the trap before the next accumulaion cycle is removed.

 

MS/MS in the ion trap

a) Ion isolation

For MS/MS purposes

, an ion is first isolated by ejecting all other ions from the trap and only fragments from the selected precursor ion will be recorded in the CID spectrum.  Mainly two methods are used to isolate the ion:

1)  Low mass ions are ejected by setting the low mass cut-off just below the mass of the ion to be isolated by raising the quadrupole amplitude.and also a fast sweep of the frequency of the dipolar amplitude on the endcaps eliminates high mass ions by resonant ejection.

2)  A wide range of frequencies are applied to the endcaps to excite and eject a broad mass range of ions.  The frequency corresponding to the parent ion is not included in the frequency range so that a small mass range is retained in the trap.

b) Ion Excitation

    To  induce fragmentation the energy of the ion of interest is increased by resonant excitation with the dipolar field , applied to the endcaps. The amplitude is below that needed for resonant ejection.   The excitaion uses a small frequency band above and below the  the precise resonance frequency to increase the stability of the excitation.  The amplitude of the excitation is less than that used for ejection and is less than 1 Volt.  The energized parent ions take up energy quickly in the dipolar field and undergo collisions with the damping gas resulting in reproducible fragmentation.  If the quadrupolar amplitude is set to high, then low mass product ions will not be trapped and if it is set to low the potential well will not be deep enough to allow the excitation of the parent ion without ejection.  The low mass cut-off is normally set to one-third of the isolation mass.  The parameters optimizing MS/MS experiments are

1. the low mass cut-off controlling the depth of the potential well.

2. the parent ion isolation time

3. the parent ion excitation time

In ion traps, the parent ions are activated  by several collisions with the background gas  but after fragmentation little energy remains in the product ions preventing further fragmentation.  By reason of the fact that  the product ions are not brought into resonance by the dipolar frequency, the MS/MS spectrum is the result of a single stage of excitation.  A second step of fragmentation without isolation is possible and this second step fragmebts fragments the first stable fragment ion to produce structural significant informations.  For an MS/MS scan the ion accumulation is identical to the steps in the MS scan.  Additional steps are introduced for isilating and fragmenting a particular ion following the accumulation of the set mass range.  First the selected m/z value is isolated by raising the quadrupolar amplitude and with a low dipolar amplitude the isolated ioin is energized to give CID without ejection while the quadrupolar amplitude is set at a level to retain the lower mass fragment ions and then a normal scan produces the fragment ion spectrum.

MSn in the Ion Trap

Instead of directly detecting the fragments, subsequent steps of isolation and fragmentation of the initial ions can be chosen for MSn spectra.  By reason of the high efficiency trapping of product associated with the ion trap, a very high percentage of ions are conserved between each stage of MS/MS and consequently the trap is a powerfull tool for structural elucidation. 

Quadrupole ion trap instrument with an external ESI ion source

 

 

 

 

 

 

 

2.4.2  MS/MS spectra of immunosuppressive drugs (Data Dr Lhost G. J. J.)

FK506 or tacrolimus is metabolized by the liver and intestinal cytochrome P-450 3A-dependent mixed-function oxygenase enzymic system to several metabolites including O-demethylated, hydroxylated, O-demethylated hydroxylated and dihydrodiol metabiltes.  The MS/MS spectrum of the FK506 C19-C20 epoxide metabolite is discussed herunder.

                 

                Electrospray MS/MS spectrum of the sodium adduct m/z = 842  (Bruker, Bremen Germany)

Fragmentation pathways of

the C19-C20 epoxide of FK506

 Electrospray MS/MS spectrum of m/z = 824

Full ESI mass spectrum (Jeol Lcmate instrument) of the FK506 C19-C20 epoxide where lthe elimination of 111 Daltons from the sodium adduct (M + Na)+ = 842 to produce a fragment ion at m/z = 731 is clearly observed.  The loss of 111 Daltons is also observed in the full mass spectrum of an ion trap instrument ((Bruker, Bremen, Germany)

 

 

 

 

 

 

 

 

 

 

 

Orbitrap

An orbitrap is a device where ions are electrostatically trapped in an orbit around a central spindle shaped electrode.  The ions both orbit around the central electrode and oscillate back and forth along the central electrode's long axis.   An image current is generated in the detector plates resulting from this oscillation.  The frequencies of these image currents depend on the mass to charge ratios of the ions.  Mass spectra are obtained by Fourier transformation of the recorded image current.

See Makarov's asms lecture      

 

2.5  Fourier Transform Ion Cyclotron Spectroscopy (FTICR)          FTICR 

The force exerted on ions placed in an area exposed to constant and uniform magnetic fields moves those ions in a circular orbit that is perpendicular to the direction of the magnetic field.  An ion cyclotron resonance (ICR) analyzer is a cubic cell consisting of two opposite                  

Fourier Transform Ion-Cyclotron Resonance Analyzer

plates, two opposite excitation plates and two opposite receiver plates.  The cell is placed between the poles of an electromagnet or superconducting solenois magnet with typical field strength in the 3 to 7 T range.  Ions formed by currently used ion source become trapped upon introduction into the cell and move in circular orbits.  The frequency of the circular motion corresponding to the cyclotron frequency f  is directly proportional to the strength of the magnetic field B and inversely proportional to the m/z ratio of the ions according to the equation

 

where f is frequency,  v  is ion velocity, r is the radius of the orbiting cycle and k is a proportionality constant.   When an rf field is superimposed  perpendicular to the direction of the magnetic field , those ions with cyclotron frequencies equal to the excitation frequency will absorb energy from the rf field and move into orbits of larger radii.  These ions are translationally excited and move in phase with the exciting field between the receiver plates.  When an external conducting network is attached to the receiving plates, the ions transmit a complex rf signal that contains frequency components related to their m/z values.  When a group of moving positive ions approaches one of the receiver plates the ions attract electrons thus creating a current and as they continue moving on their orbit and approach the other plate, they again attract electrons.  The image current signal begins to decay as the coherency is disturbed over time.  The transient free induction decay (FID) is recorded and the complex FID caused by suoerimposition of many single frequencies is deconvoluted to reveal the single contributing frequencies and their respective amplitudes.  Thus the complex time-domain image currents  produced can be transformed into frequency-domain signals by Fourier transform analysis (FT) to yield the component frequencies of the different ions from which mass spectra can be obtained using the equation given above. 

Fourier transformation: How longer the detection interval the more accurate the result.

Advantages of FTICR-MS are:

a)  By reason of the fact that ions give rise to a detectable image charge during each detector passage the sensitivity is improved.

b) ion detection is non-destructive giving the opportunity to perform MS/MS experimrnts.

c) Recording FID during a longer time allows for a precise determination of all the cyclotron frequencies yielding highest values of resolution and accurate mass determination.

 

 

 

There are four steps common to all FTICR experiments::

a) a large electric field gradient is established between the trap plates and all trapped charged particles are removed from the cell.

b) ions are injected from an external source or sample is ionized within the cell

c) ion excitation

d) ions are detected by image current

Mass scanning can be accomplished by varying the rf pulses corresponding to an irradiation frequency at a fixed magnetic field.  Because of the low drift drift velocity of the ions and the long cycloidal path, the actual times it takes for the ions to traverse the analyzer is 5 to 10 ms in contrast to the few s flight times in other analyzer types.  The low drift velocity necessitates operation in very low vacuum in the 10-10 torr region.  The long flight times of ions mean that a gas pulse raising the pressure to 10-6 torr is typical for CID experiments (the nature of the gas may be varied).  This amount of gas is readily pumped out of the cell making these instruments suited for MSn experiments.

 

2.6  Mass Spectrometry/Mass Spectrometry (MS/MS)  

2.6.1  Ion Activation Methods

The process involved in MS/MS is the fragmentation of a precursor ion into a smaller product ion accompanied by the loss of a neutral fragment.

    m+ precursor     m+ product  + N

There are several ways to provide energy i order to fragment the ions between the source and the analyzer.  The energy level acquired and its distribution within an activated ion influences the type and how much products ions are formed.  Small energy distribution favors simple bond cleavages and broad energy internal energy distribution gives rise to increased fragmentation.

2.6.1.1   Metastable Ions

When molecular ionization occurs,  the molecular ions formed remains as such or decomposes immediately (life time < 10-7 s )  into fragment ions while still in the ion source.  Most ions leaving the ion source travel unchanged through the mass analyzer to the detector.  The possibilitty exists that some travelling ions undergo unimolecular decomposition 1 to 100 s after leaving the ion source and ions undergoing such a transition are called metastable ions.  In simple focusing magnetic instruments metastable ions may form in the field free region between the ion source ane the magnetic field while in double focusing instruments there several field-free areas for metastable ion formation to occur.  Metastable ions are detected as wide Gaussian-shaped peaks of low abundance appearing as nonintegral masses included in the normal mass spectrum.  Also metastable transitions may be detected in reflector TOF instruments when a retarding potential is applied at the end of the drift tube.

2.6.1.2   Collision-Induced Dissociation

When ions with a certain high amount of translational energy are permitted to collide with a gas of high ionization potential (argon, xenon), maintained in a collision cell, a portion of the ion kinetic energy acquired during acceleration is converted into excess internal vibrational and/or electronic energy.  This process is called collisional activation.  If bonds are broken due to an excess of energy, the ions undergo collision-induced dissociation (CID) or collisionally activated decomposition (CAD). 

Low-energy collisions  :   when the kinetic energies of the ions are in the range of 1 to 100 eV, vibrational states of an ion are excited leading to narrow energy distribution and giving rise to a limited variety and quantity of product ions.  The nature and the type of fragmentation depend strongly on the collisional energy employed and on the type and pressure of the collisional gas.  Low-energy collisions can be carried out in triple quadrupole or hybrid instruments.

High-energy collisionswhen the precursor ions are accelerated to approximately 1 kV, the high-energy collision occuring with helium excite the electronic state of the analyte and produce a broad distribution of acquired internal vibational energies resulting in significant fragmentation and in structural information.  Spectra obtained are more reproducible than those obtained by low-energy CID.  High-energy collisions can be carried out in multisector magnetic or hybid instruments.

2.6.1.3  Electron-Capture Dissociation

Trapped positive ions in FTICR mass spectrometry may fragment upon encountering a high-current beam of near-zero energy electrons and ion activation may result from ion-electron collisions.  When multiply charged cations formed from ESI undergo electron capture dissociation, a specific fragmentation of the N-Ca bonds is observed in peptides and proteins providing information complementary to CID and photodissociation enabling sometimes de novo sequencing.

 

2.6.1.4  Photodissociation and Surface-Induced Dissociation.

Molecules containing a chromophore and absorbing light at a given wavelength may undergo electronic excitation upon irradiation with photons with well-defined energy (UV laser beams).  The activated ions are those absorbing at the wavelength ot the irradiating laser and as a consequence photodissociation is very selective.  A tandem TOF instrument was developed in which a linear TOF analyzer  was used for primary mass separation and precursor ion selection.  The selected ions were irradiated with a high energy laser to induce photodissociation and the product ions were analyzed with a second orthogonal acceleration reflectron TOF analyzer.

 

2.6.1.5  In-Source Fragmentation

           JEOL Lcmate ESI Source

The cone voltage may be increased in ESI sources resulting in significant fragmentation of the of the molecules under investigation giving rise to protonated molecules and abundant fragment ions.  The  main advantages of this method are:

a) the possibility to obtain MS/MS -like spectra without tandem analyzer using a single quadrupole or an oa-TOF instrument.

b) the method provides intense fragments for subsequent second generation CID in tandem instruments.

c) High sensitivity and no loss of resolution

The technique is much less specific than CID and not easily applicable to mixtures.

 

2.6.2  TANDEM-IN-SPACE MASS SPECTROMETRY

Different modes of scanning methods in MS/MS are reported briefly herunder: (Example of a QqQ instrument)

2.6.2.1  Product-Ion Scanning Mode

A selected ion is focused through the first quadrupole and reacted with the neutral target gas of the second quadrupole working as a collision cell.  The ions formed by CID are separated according to their m/z values in the third quadrupole.  A product ion mass spectrum is obtained derived from the selected precursor ion.  This type of MS/MS experiment is the most frequently used providing informations about the structure of the selected precursor ion.

2.6.2.2  Precursor Scanning Mode

Ions are mass separated in the first quadrupole and are submitted to CID in the second quadrupole and  a preselcted mass is allowed to pass in the third quadrupole mass filter.  This type of experiment is producing a precursor ion spectrum which dissociate to give a specific product ion.

2.6.2.3  Constant Neutral Loss Scanning Mode

The first and the third quadrupole are scanned at the same time with a predefined mass offset.  Specific informations about the mass losses occuring during the CID process in the collision cell of the second quadrupole are obtained.

2.6.2.4  Selected Reaction Monitoring  (SRM)

In the SRM mode, a preselected precursor is introduced in the collision cell and only some specific product ions are selected for detection the other product ions being rejected.  This type of experiment may be compared to selected ion monitoring (SIM) in single analyzer instruments.

2.6.2.5  Post-Source Decay (Time of flight)

Reflectrons (Ion Mirrors)

While delayed extraction is an in-source method of energy compensation, the ion mirror is a postsource  compensation method for energy inhomogeneity.   The resolution of TOF analyzers is limited for several reasons including :

1. as the mass of the analyte increases , the difference in arrival times of ions at the detector becomes smaller and smaller, and more difficult to differentiate.

2. there is a spread in the temporal, spatial and kinetic energy distribution of the groups of ions of the same mass as they are are pulsed out of the source into the flight tube. This means that two ions of the same mass formed at different times, or at different locations in the source, will arrive at the detector at different times.

Products of post-source metastable fragmentation cannot be distinguished with linear TOF analyzers by reason of the fact that precursor and product ions move with the same velocity and arrive simultaneously at the detector.  Reflector TOF analyzers separate precursor and post-source decay (PSD) products by their difference in kinetic energy in the ion mirror.

 

 

    REFLECTOR TOF ANALYZER

 

 

 

 

 

 

 

 

 

 

 

2.6.3   TANDEM-IN-TIME MASS ANALYZERS

The advantagess of trapped-ion analyzers     in MS/MS include the possibility to conduct MSn experiments and particularly in the case of FTICR                high resolution is another advantage.