LTQ FTICR

Table 5.2 shows a representative set of accurate mass measurements of 50 pharmaceutical compounds which were acquired using an eight-scan event experiment on the LTQ-FTICR (Josephs et al., 2004b). Mass spectra from all eight scan events were acquired within 7 s, the width (at the base) of a typical chromatographic peak, as shown in Fig. 5.3 for warfarin (MW 308). The first scan event is a full-scan positive-ion MS acquired in the ion trap. If the expected protonated molecule is detected above a preset threshold, a second scan event, an ion trap (IT) data-dependent MS/MS scan, is triggered. This is followed by a third scan event, where the protonated molecule is accurately measured in the FT, and a fourth scan event, a full-scan MS/MS in the FT. 

Figure 5.4. Schematic of a hybrid LTQ-FTICR illustrating the distance between the ion trap and the ICR cell. Shown is an expansion of the ion transfer optics and how ions separate on their way to the ICR cell and how the low-mass ions could enter the cell, be reflected at the rear of the cell, and exit the cell while waiting for the higher mass ions to enter the cell. (Courtesy of ThennoFisher Scientific.)...

The LTQ-Orbitrap has resolution and mass accuracy performance close to that of the LTQ-FTICR. As shown in Table 5.3 (column 4), LTQ-Orbitrap accurate mass measurements, using external calibration, for a set of 30 pharmaceutical compounds resulted in less than 2.3 ppm error. The data were acquired with a 4-min, 1-mL/min-flow-rate, positive-mode LC-ESI-MS method where all measurements were performed within 5h from mass calibration. Mass accuracies below 2-3 ppm, and often below 1 ppm, can be routinely achieved in both the positive- and negative-ion mode (Table 5.3, columns 4 and 5). The long-term mass stability of the LTQ-Orbitrap is not as consistent as observed for the LTQ-FTICR-MS, and the Orbitrap requires more frequent mass calibration however, mass calibration is a routine procedure that can be accomplished within 5-10 min. Figure 5.7 displays a 70-h (external calibration) mass accuracy plot for three negative ions collected with a LTQ-Orbitrap where the observed accuracy is 2.5 ppm or better with little mass drift for each ion. Overall, for routine accurate mass measurements on the Orbitrap, once-a-week calibration (for the desired polarity) is required however, considering the ease of the process, more frequent external calibration is not a burden. 

Although both hybrid LTQ-FTMS instruments find applications in the dmg metabolism area, the LTQ-Qrbitrap, with its simplified maintenance, lower cost, and in most cases better sensitivity at low masses, is generally more applicable to small-molecule analysis. The LTQ-FTICR, with the advantage of higher resolution and mass accuracy, covers a wider range of applications, from small molecules to peptides and proteins, where most advantages are found in the analysis of large biomolecules. 

The full-scan BPC obtained from LTQ-FTICR LC-MS data does not display diclofenac or its monohydroxyl metabolite (Ml) that were spiked into rat bile sample due to the significant interference from ions of endogenous components in the bile (Fig. 6.9a). In contrast, MDF analysis of the LC-MS data file completely removed these interfering ions, resulting in a BPC that clearly exhibits only the drug and Ml (Fig. 6.9b). The same bile sample was analyzed with NLS and PIS using a triple-quadmpole instrument. Diclofenac lost a neutral fragment (46 Da) to form a major ion at m/z 250 under CID conditions (Fig. 6.8a). 

Figure 6.9. Detection of diclofenac metabolite in rat bile by three LC-MS screening methods (a) full-scan BPC from LTQ-FTICR (b) MDF-treated full-scan BPC from LTQ-FTICR (c) ion current of NLS of 46 Da (d) ion cunent of PIS of m/z 250 (e) ion current of PIS of m/z 266.

Another example is the analyses of the clozapine metabolites M2 (the N-desmethyl product) and M3 (N-oxide) in rat urine. The metabolite peaks of M2 and M3 were hardly observable in the full-scan TIC obtained by LC-LTQ-FTICR (Fig. 6.10a). After removing the majority of endogenous components and chemical noise by MDF, M2, M3, and clozapine became the predominant peaks in the full-scan TIC (Fig. 6.1 Oh). Precursor ion scan analysis of the same spiked... 

Both of the above examples illustrate that the sensitivity of the MDF method, in general, is comparable to PIS analysis and better than NLS methods (Figs 6.9 and 6.10). The sensitivity of the MDF method is attributed at least in part to the full-scan capability of high-resolution mass spectrometers such as the Q-TOF and LTQ-FTICR, which could be several times more sensitive than full-scan analysis by a triple-quadrupole mass spectrometer (Kostiainen et al., 2003). 

Figure 6.12. Base peak chromatograms obtained from 75-/xL urine samples spiked with clozapine metabolites using LTQ-FTICR. (a,b) Samples spiked with oxidative metabolites, where (a) was from unprocessed data and (b) from MDF-processed data. (c,d) Samples spiked with GSH metabolites, where (c) was from unprocessed data and (dj from MDF-processed data. The MDF settings were 0.04 Da in mass defect dimension and 50 Da around the parent or the parent-plus-GSH mass, respectively.

Nevertheless, even with accurate mass measurement by Q-TOF, LTQ-Orbitrap, or LTQ-Fourier transform ion cyclotron resonance (LTQ-FTICR) MS, it is not always possible to fully characterize certain metabolites based solely on mass spectrometric... 

Commercial LITs were introduced in 2002 as either a stand-alone mass spectrometer (LTQ) [318] or as part of a triple quadrupole (Q-Trap) [319] or in 2005 as part of hybrid tandem mass spectrometers (LTQ-Orbitrap and LTQ-FTICR) [88,90], Application of LTQ-FTICR for metabolism studies has been reviewed by Shipkova et al. [90], In comparison to other mass analyzer types, FTICR-based mass spectrometers are not very popular for metabolite identification studies due to availability of less expensive and more user-friendly LTQ-Orbitrap and Q-TOF-based systems. Another limitation associated with the FTICR-based hybrid mass spectrometers is the TOF effect, which results in efficient trapping of only the high-mass ions [90],... 

Triple quadrupole, Q-TOF, QIT, LIT, FTICR, Q-FTICR, LTQ-orbitrap, and LTQ-FTICR. 

FIGURE 13.3 Instrument schematics for (a), Waters Synapt QTOF ( Waters), (b) Thermo Finnigan LTQ-Orbitrap, and (c) Thermo Finnegan LTQ FTICR mass spectrometers ( Thermo Finnegan). 

Hybrid linear ion trap-Fourier transform ion cyclotron resonance (FTICR) mass spectrometer, 7T (LTQ-FTICR,Thermo), or equivalent instrument, equipped with an electrospray/nanospray (ESI/ NSI) source and ECD accessory, operated under the control of Xcalibur software package (Thermo Fisher Scientific, San Jose, CA Instrument Configuration, Instrument Setup, Sequence Setup, and Qual Browser modules appropriately created and used) with Bioworks available to preprocess raw data files for database search. ECD may be replaced with ETD and the respective manufacturer s own version of the above software should be used, when alternative instrument is selected. 

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