Hybrid MS instruments including TOF analyzers Due to their high transmission range, resolution and accuracy, TOF analyzers have been implemented, in conjunction with a different mass analyzer, in a number of hybrid mass spectrometers, usually having MS/MS or MSn capabilities. The most common couplings are: Quadrupole Ion Trap – TOF (QIT-TOF) Quadrupole – TOF (Q-TOF) Both MALDI and ESI/APCI (API) have been used as ionization sources for these hybrid spectrometers.

MALDI-QIT-TOF hybrid MS In this system the ion beam resulting from the MALDI process is directed to a quadupole ion trap and accumulated. Decoupling of the ionization process from the mass measurement is thus achieved. Indeed, the ion trap is used to cool down all ions (through helium collisions) and locate them at a very well specified point in space with minimal kinetic energy before ejection for TOF measurement. During MS/MS and MSn experiments precursor ions are isolated and fragmented into the QIT, then product ions are ejected and analyzed by the TOF analyzer.

The nature and pressure of the buffer gas within the ion trap has a significant influence on mass resolution, due to different ion cooling efficiency. The effect on the mass spectrum of the [M+6H]6+ ion from bovine insuline is clearly shown: 956.6036 (-6.5 ppm) 1 m/z

Mass accuracy of 3-10 ppm is usually achieved and is independent from ionization conditions, sample, matrix type and laser power. Sensitivity is in the low femtomole (10-15 moles) range.

MALDI-QIT-TOF: a bioanalytical application The Shimadzu Axima QIT-ToF spectrometer has been used for the MS and MS/MS analysis of protein-related bidimensional polyacrylamide gel electrophoresys (2D-PAGE) spots after in-gel digestion with trypsine. Sample preparation: transfer of protein spots from Escherichia coli, separated by 2D-PAGE; on-spot enzymatic digestion by trypsin using a chemical printer to dispense the enzyme and other digestion reactants; micro-dispensing of MALDI matrix on spots previously subject to trypsin digestion.

MS spectra MS/MS spectra

LC-ESI-QIT–TOF hybrid MS This hybrid instrument is similar to MALDI-QIT-TOF, at least in principle. The only, though significant, difference is that ions are continuously transferred by the ESI source (whereas MALDI is a pulsed ion source). Ion optics preceding the QIT are then required to pulse the ion beam entering the trap. In the Shimadzu LC-MS-IT-TOF this function is effected by the combination of a skimmer, a octopole and a single lens assembly: CDL : Curved Desolvation Line

MS/MS and MSn experiments can be performed, as usual, in the 3D-ion trap of the LC-MS-IT-TOF. In the Shimadzu instrument a new technology is adopted for precursor ion fragmentation through CID. Indeed, the buffer gas, Argon, is pulsed through a valve into the trap just before a CID experiment. Ar pulsing improves the dependence of the signal related to product ions (purple points) on excitation voltage amplitude, thus increasing the S/N ratio of MS/MS and MSn spectra:

Ion transfer from the 3D-ion trap to the TOF analyzer must occur in a very short time, to avoid excessive temporal spreading. In the Shimadzu LCMS-IT-TOF the transfer is achieved through a technology known as Ballistic Ion Extraction (BIE): When the ions to be transferred (precursor or product ions) are ready inside the trap the ring electrode is grounded while a high dc voltage pulse is applied between the end caps so that ions are pushed towards the TOF analyzer.

Analysis of a mixture of drugs by LC-ESI-IT-TOF-MS:

Collisional focusing quadrupole ion guide MALDI-Q-TOF Hybrid MS (with orthogonal injection) In this system a quadrupole ion guide focuses ions that are coming off the source, thus reducing their energy distribution to improve resolution and sensitivity: Ions exiting the quadrupole are transferred to the TOF analyzer by orthogonal injection, a technique developed already in the 1960s but introduced in commercial spectrometers only 30 years later. Orthogonal injection is effected by a device known as Ion modulator or Pusher. laser TOF Collisional focusing quadrupole ion guide

Ion modulator/pusher VTOF Ions exiting the quadrupole are transferred by ion optics in a storage region, limited by a plate and a grid (G1), that are initially kept at the same potential (0 V). The ions can move along their original direction in this field-free region. Push-out pulse storage region plate ions kHz frequency G1 VTOF G2 Afterwards, an injection (push-out) voltage pulse is applied to the plate: the ions are thus pushed in the orthogonal direction. Once passed through grid 1, they are subject to a VTOF voltage, applied to grid 2 (G2), pushing them inside the TOF analyzer. When all ions have moved to the drift region of the TOF analyzer, the voltage on the plate and on Grid 1 is restored to 0 V and ions from the ion source begin to fill the storage region again.

VTOF plate G1 G2 ions It is worth noting that orthogonal acceleration gives ion velocity a new component (blue vector), normal to that related to their initial velocity (red vector). The resulting drift trajectories are then inclined (green vector). Ions entering the pusher must have a limited energy spread, otherwise a spread in drift trajectories will be observed.

RF-only or mass selecting quadrupole (RP = 1000-2000) LC-ESI-Q(q)TOF hybrid MS Orthogonal injection is the best way to couple continuous ion sources (like API ones) with a TOF analyzer. This approach has been applied in hybrid quadrupole-TOF (Q-TOF) spectrometers, having also MS/MS capabilities, due to the presence of a collision cell. The Qstar series spectrometers, from Applied Biosystems, are an example: RF-only quadrupoles RF-only or mass selecting quadrupole (RP = 1000-2000) Reflectron-TOF (RP = 10000) Note standard lockspray source T-WAVE ion guide Quadrupole pDRE lens located in front of collision cell T-WAVE collision cell New design of transfer optics after collision cell Modular Tof head equipped oa-Tof with V and W optics

In a Q(q)-TOF instrument pressure needs to be carefully controlled in each region (by combining the size of apertures joining adjacent regions and the pumping speed):

The presence of a significant gas pressure (10 mTorr) in quadrupoles Q0 and Q2, due, respectively, to a mixture of air and ESI solvents vapors and to purposely introduced Argon, is fundamental to ensure collisional dampening in Q0 (i.e. reduction of the initial energetic and spatial spread of ions) and CID of precursor ions (selected by Q1) – collisional dampening of product ions in Q2 The mean free path for ions at 10 mTorr is ca. 5 mm, thus each ion is typically involved in 10-100 collisions in Q0 and Q2.

Importance of ion kinetic energies in a Q(q)TOF spectrometer When leaving the Q0 ions possess low (thermal) kinetic energies (< 10 eV). Their fate in the subsequent regions of the spectrometer, related to the chosen analysis mode, is determined by acceleration potentials adopted between the quadrupoles (offset voltages OV1 and OV2): OV1 OV2 MS Mode OV1 = 10-30 V, Q1 in RF-only mode, OV2 = 10-20 V: ions are thermalized and focused on the central axis but not fragmented CID-MS/MS Mode OV1 = 10-30 V, Q1 in mass selective mode, OV2 = 100-300 V: CID occurs in the first part of Q2, then thermalization of product ions occurs

Duty cycle of a Q(q)TOF spectrometer In a Q(q)TOF spectrometer only ions present in the storage region of the Ion Modulator/Pusher are actually pushed to the TOF when a pulse is applied: All the other ions are lost, thus reducing the Duty Cycle of Q(q)TOF instruments to 5-30%. Depending on the m/z range of interest, pulse frequencies vary between 5 and 15 kHz. For a 10 kHz frequency a inter-pulse time of 0.1 ms is adopted, being approximatively the time required for the highest m/z ions to arrive at the detector.

Double reflectron-LC-ESI-Q-TOF hybrid MS A more complex Q(q)TOF instrumentation is represented by the Waters-Micromass QToF series. In the Q-ToF Ultima/Micro instrument the TOF analyzer is equipped with a second reflectron and the collision cell is a hexapole: Second reflectron

The highest resolving power (about 17000) is obtained when the second reflectron is active, i.e. in the so-called W mode, usually adopted for special MS measurements: In this case the first quadrupole and the collision cell are used as ion bridges, i.e. in the RF-only mode, to transfer ions incoming from the ESI source to the TOF analyzer.

When MS/MS measurements are required, only one reflectron is usually active, thus the TOF is operated in the V-mode, with a maximum resolving power about 10000: In this case the quadrupole operates in mass selective scan mode and the hexapole as a collision cell, in which precursor ions, incoming from the quadrupole and accelerated by an offset voltage, undergo CID against Argon atoms.

A Z-spray ESI-source is usually mounted on the Micromass spectrometers: In order to enhance ESI efficiency, in the Z-spray source ion sampling occurs in an orthogonal geometry and is assisted by a cone gas, that effects desolvation before ions enter the ion block.

Lockspray source The Lockspray is a special ESI source providing the capability to introduce a reference compound through a different electrospray probe. A baffle oscillating at a user-defined frequency allows alternate analysis of sample and reference spray. m/z drifts naturally occuring in TOF analyzers (i.e. due to flight tube micro-deformations induced by laboratory temperature modifications) can be corrected using the m/z value(s) of the reference compound. Mass accuracies lower than 5 ppm can be usually achieved. The in-source introduction of the reference compound avoids interferences with the LC separation of analytes.