Q-TOF instruments

The Q in Q/TOF stands for quadrupole (see Chapter 25, Quadrupole Ion Optics ). A Q/TOF instrument — normally used with an electrospray ion inlet — measures mass spectra directly to obtain molecular or quasi-molecular mass information, or it can be switched rapidly to MS/MS mode to examine structural features of ions. The analyzer layout is presented in Figure 20.2. 

The term Q/TOF is used to describe a type of hybrid mass spectrometer system in which a quadrupole analyzer (Q) is used in conjunction with a time-of-flight analyzer (TOP). The use of two analyzers together (hybridized) provides distinct advantages that cannot be achieved by either analyzer individually. In the Q/TOF, the quadrupole is used in one of two modes to select the ions to be examined, and the TOF analyzer measures the actual mass spectrum. Hexapole assemblies are also used to help collimate the ion beams. The hybrid orthogonal Q/TOF instrument is illustrated in Figure 23.1. 

Schematic diagram of an orthogonal Q/TOF instrument. In this example, an ion beam is produced by electrospray ionization. The solution can be an effluent from a liquid chromatography column or simply a solution of an analyte. The sampling cone and the skimmer help to separate analyte ions from solvent, The RF hexapoles cannot separate ions according to m/z values and are instead used to help confine the ions into a narrow beam. The quadrupole can be made to operate in two modes. In one (wide band-pass mode), all of the ion beam passes through. In the other (narrow band-pass mode), only ions selected according to m/z value are allowed through. In narrow band-pass mode, the gas pressure in the middle hexapole is increased so that ions selected in the quadrupole are caused to fragment following collisions with gas molecules. In both modes, the TOF analyzer is used to produce the final mass spectrum.

Chapter 23 Hybrid Quadrupole Time-of-Flight (Q/TOF) Instruments... 

As with the Q-ToF instrument, only two types of MS-MS experiment are available with the ion-trap, i.e. the product-ion scan and selected-decomposition monitoring, as described in Sections 3.4.2.1 and 3.4.2.4, respectively. 

The ion-trap and Q-ToF instruments are, because of the way that they operate, unable to carry out precursor-ion scans. Computer manipulation of data generated during product-ion scans of the Q-ToF system, however, can yield equivalent data to that produced directly by precursor-ion scans on other instruments and an evaluation of this software-based approach has been carried out [14],... 

The structure of Bosentan [30] and three of its metabolites are shown in Figure 5.45 and the product-ion spectra from the [M - - H]+ ions from these compounds in Figure 5.46. All show an ion at m/z 280 which might be assumed, simplistically, to share the same structure. Their accurate masses, determined by using a Q-ToF instrument, however, show that the ions from compounds (1)... 

Rindgen, D., Cox, K., Clarke, N. and Korfmacher, W., An Integrated Approach to Metabolite Identification for the Drug Discovery Compound SCH 123 using the Triple Quadrupole, Ion Trap and Q-TOF Instruments, American Society for Mass Spectrometry 2000 Conference Abstract, Long Beach, CA, USA, 2000. 

A Q-TOF spectrometer is similar to a triple quadrupole but Q3 is replaced by an orthogonal TOF mass spectrometer. Using a Q-TOF instrument only the product ion scan mode can be collected, but because of its high resolving power, accurate masses for both the precursor ion and product ions can be obtained. (See the section below on accurate mass measurements.)... 

Over the years, the Q-TOF instrumentation has become an important mass spectrometer of choice for scientists working in the drug metabolism arena. This is due to the fact that Q-TOF offers very high sensitivity in the full-scan MS and... 

The resolution at low masses is less than the one obtained with FTICR. However, the resolution of FTICR is inversely proportional to m/z whereas the resolution of the orbitrap is inversely proportional to m/z)112 and thus decreases more slowly when the mass increases. Compared with a Q-TOF instrument, the resolution is dramatically increased as well as the dynamic range. 

Many instrumental set-ups and geometries have been explored. In triple quadrupole mass spectrometry, the first quadrupole selects the parent ion of interest, the second works as a collision cell to fragment the parent ion, and the third isolates the proper product ion. A hybrid type is the quadrupole time-of-flight (Q-TOF) instrument. 

Ion-trap MS-MS spectra for paraquat, diquat, difenzoquat, mepiquat, and chlormequat were reported [60-61]. The fragmentation pathways were discussed in considerable detail [60]. The interpretation of the product-ion MS-MS spectra was checked and studied in more detail using accurate product-ion determination via MS-MS on a Q-TOF instrument. The elucidation of the fragments observed for paraquat, diquat, mepiquat, chlormequat, and difenzoquat was tabulated [62]. 

In order to take full advantage of the enhanced sensitivity of nano-ESI (Ch. 5.5.5), and the ability to perform spectrum averaging and multiple MS-MS experiments on Q-LIT and Q-TOF instruments, Staack et al. [19] proposed RPLC fractionation into 20 fractions, which are subsequently analysed by a commercial microchip-nano-ESI system at 200 nl/min. 

TOF and Q-TOF instraments are freqnently applied in metabolism studies. The identity of the epothilone B metabolites fonnd by precnrsor-ion analysis (Figure 10.5, Ch. 10.4.2) was confirmed using accnrate-mass determination on a LC-TOF-MS instmment [29]. Some other examples are the characterization of metabolites of moclobemide and remikiren [32], the identification of ketobemidone Phase-I and Phase-II metabolites [33], and the identification of in vitro metabolites of ethoxidine [34]. The nse of a five-channel multiplexed ESI interface (four chaimels for parallel LC-MS and one channel for lock-mass componnd infusion) on a Q-TOF instrument was recently described to speed np metabolite identification and to enhance the efficient nse of the costly instrument [35]. 

In a series of papers, the group of Volmer [130-132] studied the analysis of azaspiracid biotoxins. Ultrafast and/or high-resolution LC of azaspiracids on monohthic LC columns was evaluated [130]. Chromatograms of five azaspiracids on a 100-mm and a 700-mm monolithic column are shown in Figure 14.11. Fragmentation of azaspiracids in MS-MS on ion-trap and triple-quadrupole instruments was studied as well [131]. The interpretation was confirmed using accurate-mass data from a Q-TOF instrument. Validation of a quantitative method for AZA-1 was also reported [132]. The LOQ was 5 and 50 pg/ml extract using a triple-quadrapole in SRM mode and an ion-trap instrument, respectively. 

March and Miao [16] performed a detailed study on the fragmentation of the flavonol kaempferol, using aeertrate-mass determination with a Q-TOF instrument. The data were consistent with previous findings. Next to the common C-ring fragmentations, leading to + 2H and the pairs and - 2H, and ° B and losses of CjHjO, CHO, CO, and HjO were observed. 

Using accurate-mass determination with a Q-TOF instrument, March and Miao [16] studied the fragmentation of deprotonated kaempferol. The fragments observed were apparently not consistent with the above discussion. Accurate-mass assisted interpretation indicated that most fragments can be explained from consecutive losses of small neutrals, especially HjO, OH , CO, CHjO, and CjHjO. 

Zhou and Johnston [55] reported protein characterization by capillary isoelectric focussing (CIEF) on-hne coupled to RPLC-MS. Direct coupling of CIEF to ESl-MS is limited by interferences by the ampholytes. Inserting RPLC in-between can help removing these interferences. CIEF is performed in combination with a microdialysis membrane-based cathodic cell to remove the ampholyte and to collect protein fractions by stop-and-go CIEF prior to transfer to a 5><0.3-pm-ID C,8 trapping colunm and RPLC separation on a 50><0.3-pm-ID C4 column. The separation is performed using an acetonitrile-water gradient (0.1% acetic acid). ESI-MS is performed on a quadrupole-TOF hybrid (Q-TOF) instrument. 

A microchip device with an attached nano-ESl emitter tip was developed to facilitate the introduction of tiyptic digests by means of nano-ESl [88-89]. Instead of off-line filling of the nano-ESl needle, the sample is transferred from a vial on the chip to the nano-ESl needle by electroosmosis. Detection limits of 2 fmol/pl were achieved for fibrinopeptide A (1699 Da). Further developments enabled sequential automated analysis of protein digests by ESl-MS [90]. On-chip sample pretreatment and desalting by either sample stacking via polarity switching or SPE prior to on-chip CE was described by Li et al. [91], and applied to the identification of 2D-GE separated proteins from Haemophilus influenzae using a Q-TOF instrument. 

Data-dependent switching between a survey-MS mode and the product-ion MS-MS mode (Ch. 2.4.2) in the LC-MS analysis of tryptic digest on a triple-quadrapole instrument was pioneered by Stahl et al. [140]. The MS-MS spectra obtained were correlated wiA a protein sequence database by using the SEQUEST program. DDA (also called SmartSelect, or Information-Dependent Acquisition, IDA) on ion-trap, Q-LIT, and Q-TOF instruments have become important tools in high-throughput protein characterization. 

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