Development and characterization of an electrospray ionization ion trap/linear time-of-flight mass spectrometer

On-line analysis of compounds from solution has been greatly facilitated by the advent of electrospray ionization mass spectrometry (ESI-MS). Although quadrupole mass analyzers are most commonly used with ESI at present, time-of-flight (TOF) mass spectrometers offer several potential advantages including high data acquisition rates, which are desirable for fast separation techniques. One method of coupling ESI and TOF uses an ion trap for temporary storage and accumulation of the electrosprayed ions prior to TOF mass analysis. Previous studies have not fully addressed the effects of several key variables on the analytical capabilities of this type of instrument. In this study, the characterization of an ion trap/linear TOF instrument for ESI is described. The behavior of various analytes is divided into two separate groups; each one is found to have its own optimal set of operating conditions. The reasons for the observed differences between groups are explored. Issues relevant to mass resolution, sensitivity, mass range, mass-to-charge ratio discrimination, and mass measurement accuracy are addressed. Finally, it is suggested that the analytical capability of this type of instrument could be significantly improved by changing the ion optics from the existing focusing lenses to a rf-only quadrupole lens.     

Use of a quadrupole linear ion trap mass spectrometer in metabolite identification and bioanalysis

A new type of quadrupole linear ion trap mass spectrometer, Q TRAP trade mark LC/MS/MS system (Q TRAP trade mark ), was evaluated for its performance in two studies: firstly, the in vitro metabolism of gemfibrozil in human liver microsomes, and, secondly, the quantification of propranolol in rat plasma. With the built-in information-dependent-acquisition (IDA) software, the instrument utilizes full scan MS in the ion trap mode and/or constant neutral loss scans as survey scans to trigger product ion scan (MS(2)) and MS(3) experiments to obtain structural information of drug metabolites 'on-the-fly'. Using this approach, five metabolites of gemfibrozil were detected in a single injection. This instrument combines some of the unique features of a triple quadrupole mass spectrometer, such as constant neutral loss scan, precursor ion scan and multiple reaction monitoring (MRM), together with the capability of a three-dimensional ion trap. Therefore, it becomes a powerful instrument for metabolite identification. The fast duty cycle in the ion trap mode allows the use of full product ion scan for quantification. For the quantification of propranolol, both MRM mode and full product ion scan in the ion trap mode were employed. Similar sensitivity, reproducibility and linearity values were established using these two approaches. The use of the product ion scan mode for quantification provided a convenient tool in selecting transitions for improving selectivity during the method development stage.     

A linear ion trap mass spectrometer with versatile control and data acquisition for ion/ion reactions

A linear ion trap (LIT) with electrospray ionization (ESI) for top-down protein analysis has been constructed. An independent atmospheric sampling glow discharge ionization (ASGDI) source produces reagent ions for ion/ion reactions. The device is also meant to enable a wide variety of ion/ion reaction studies. To reduce the instrument's complexity and make it available for wide dissemination, only a few simple electronics components were custom built. The instrument functions as both a reaction vessel for gas-phase ion/ion reactions and a mass spectrometer using mass-selective axial ejection. Initial results demonstrate trapping efficiency of 70% to 90% and the ability to perform proton transfer reactions on intact protein ions, including dual polarity storage reactions, transmission mode reactions, and ion parking.     

A novel approach to perform metabolite screening during the quantitative LC-MS/MS analyses of in vitro metabolic stability samples using a hybrid triple-quadrupole linear ion trap mass spectrometer

In vitro metabolic stability experiments using microsomes or other liver preparations are important components in the discovery and lead-optimization stages of compound selection in the pharmaceutical industry. Currently, liquid chromatography-tandem mass spectrometric (LC-MS/MS) support of in vitro metabolic stability studies primarily involves the monitoring of disappearance of parent compounds, using selected reaction monitoring (SRM) on triple-quadrupole instruments. If moderate to high turnover is observed, separate metabolite identification experiments are then conducted to characterize the biotransformation products. In this paper, we present a novel method to simultaneously perform metabolite screening in addition to the quantitative stability measurements, both within the same chromatographic run. This is accomplished by combining SRM and SRM-triggered, information-dependent acquisition (IDA) of MS/MS spectra on a hybrid triple-quadrupole linear ion trap (QqQLIT) mass spectrometer. Microsomal stability experiments using model compounds, bufuralol, propranolol, imipramine, midazolam, verapamil and diclofenac, were used to demonstrate the applicability of our approach. This SRM + SRM-IDA approach generated metabolic stability results similar to those obtained by conventional SRM-only approach. In addition, MS/MS spectra from potential metabolites were obtained with the enhanced product ion (EPI) scan function of LIT during the same injection. These spectra were correlated to the spectra of parent compounds to confirm the postulated structures. The time-concentration profiles of identified metabolites were also estimated from the acquired data. This approach has been successfully used to support discovery programs.     

A strategy for identification of drug metabolites from dried blood spots using triple-quadrupole/linear ion trap hybrid mass spectrometry

Discovery stage studies that address issues of absorption, distribution, metabolism and excretion (ADME) are vital for lead optimization resulting in new drug candidates. Often pharmacokinetics (PK) is assessed in these experiments without regard for the metabolism of the compound or the potential for metabolites to circulate in vivo. This work presents a strategy for drug level determination and detection of metabolites using dried blood spots for sample collection. Initially, metabolites are detected from microsomal incubations and characterized using tandem mass spectrometry. Data dependent enhanced MS and enhanced product ion (EMS-EPI) scanning with dynamic background subtraction was used on a hybrid quadruple linear ion trap mass spectrometer. On-the-fly background subtraction greatly improved the detection of metabolites. These data were used to build a multiple reaction monitoring (MRM) method for the parent and metabolites. MRM-EPI scanning was used to analyze the extracted dried blood spots from the PK study. Circulating metabolites were detected using MRM and their identities confirmed on the basis of fragment ion spectra collected simultaneously. The use of dried blood spots provides a means for re-analysis of PK samples for metabolite identification without the need for complex sample storage and preparation. Both parent compound and metabolite information can be collected in these studies, resulting in a savings of time and resources.     

Product ion scanning using a Q-q-Q linear ion trap (Q TRAP) mass spectrometer

The use of a Q-q-Q(linear ion trap) instrument to obtain product ion spectra is described. The instrument is based on the ion path of a triple quadrupole mass spectrometer with Q3 operable as either a conventional RF/DC quadrupole mass filter or a linear ion trap mass spectrometer with axial ion ejection. This unique ion optical arrangement allows de-coupling of precursor ion isolation and fragmentation from the ion trap itself. The result is a high sensitivity tandem mass spectrometer with triple quadrupole fragmentation patterns and no inherent low mass cut-off. The use of the entrance RF-only section of the instrument as accumulation ion trap while the linear ion trap mass spectrometer is scanning enhances duty cycles and results in increased sensitivities by as much as a factor of 20. The instrument is also capable of all of the triple quadrupole scans including multiple-reaction monitoring (MRM) as well as precursor and constant neutral loss scanning. The high product ion scanning sensitivity allows the recording of useful product ion spectra near the MRM limit of quantitation.     

A secondary ion microprobe ion trap mass spectrometer

An ion trap mass analyzer has been attached to an organic secondary ion microprobe. A pressure differential >100 can be maintained between the ion trap and microprobe. The well-focused secondary ion beam can transit a small (2 mm) diameter tube, but gas flow from ion trap to microprobe is impeded. This pressure differential allows the microprobe to retain imaging capability. Ion trap and microprobe data systems are integrated by taking advantage of the highly reproducible periodicity of the ion trap operating in resonant ejection mode and asynchronous signal and data acquisition afforded by commercially available interface cards. Secondary ion mass spectra and images obtained indicate an approximately 10-fold improvement in sensitivity, although preliminary evidence indicates low (<1%) trapping efficiency. Image data acquisition using the ion trap for mass analysis requires at least 10 times as much time compared to using a quadrupole mass filter because the mass-selected instability mode is used for mass analysis, i.e., mass resolution in the ion trap is not continuous as it is in the quadrupole.     

Rapid screening and characterization of drug metabolites using a multiple ion monitoring-dependent MS/MS acquisition method on a hybrid triple quadrupole-linear ion trap mass spectrometer

A novel LC/MS/MS method that uses multiple ion monitoring (MIM) as a survey scan to trigger the acquisition of enhanced product ions (EPI) on a hybrid quadrupole-linear ion trap mass spectrometer (Q TRAP) was developed for drug metabolite identification. In the MIM experiment, multiple predicted metabolite ions were monitored in both Q1 and Q3. The collision energy in Q2 was set to a low value to minimize fragmentation. Results from analyzing ritonavir metabolites in rat hepatocytes demonstrate that MIM-EPI was capable of targeting a larger number of metabolites regardless of their fragmentation and retained sensitivity and duty cycle similar to multiple reaction monitoring (MRM)-EPI. MIM-based scanning methods were shown to be particularly useful in several applications. First, MIM-EPI enabled the sensitive detection and MS/MS acquisition of up to 100 predicted metabolites. Second, MIM-MRM-EPI was better than MRM-EPI in the analysis of metabolites that undergo either predictable or unpredictable fragmentation pathways. Finally, a combination of MIM-EPI and full-scan MS (EMS), as an alternative to EMS-EPI, was well suited for routine in vitro metabolite profiling. Overall, MIM-EPI significantly enhanced the metabolite identification capability of the hybrid triple quadrupole-linear ion trap LC/MS.     

Rapid screening and characterization of drug metabolites using a new quadrupole-linear ion trap mass spectrometer

The application of a new hybrid RF/DC quadrupole-linear ion trap mass spectrometer to support drug metabolism and pharmacokinetic studies is described. The instrument is based on a quadrupole ion path and is capable of conventional tandem mass spectrometry (MS/MS) as well as several high-sensitivity ion trap MS scans using the final quadrupole as a linear ion trap. Several pharmaceutical compounds, including trocade, remikiren and tolcapone, were used to evaluate the capabilities of the system with positive and negative turbo ionspray, using either information-dependent data acquisition (IDA) or targeted analysis for the screening, identification and quantification of metabolites. Owing to the MS/MS in-space configuration, quadrupole-like CID spectra with ion trap sensitivity can be obtained without the classical low mass cutoff of 3D ion traps. The system also has MS(3) capability which allows fragmentation cascades to be followed. The combination of constant neutral loss or precursor ion scan with the enhanced product ion scan was found to be very selective for identifying metabolites at the picogram level in very complex matrices. Owing to the very high cycle time and, depending on the mass range, up to eight different MS experiments could be performed simultaneously without compromising chromatographic performance. Targeted product ion analysis was found to be complementary to IDA, in particular for very low concentrations. Comparable sensitivity was found in enhanced product ion scan and selected reaction monitoring modes. The instrument is particularly suitable for both qualitative and quantitative analysis.     

Analysis of high mass peptides using a novel matrix-assisted laser desorption/ionisation quadrupole ion trap time-of-flight mass spectrometer

A novel matrix-assisted laser desorption/ionisation quadrupole ion trap time-of-flight (MALDI QIT ToF) mass spectrometer has been used to analyse high mass peptide ions exceeding 2000 Da. Human adrenocorticotropic hormone (fragment 18-39) and oxidised bovine insulin chain B were utilised to evaluate the performance of the instrument both in MS and in MS/MS mode. Its ability to efficiently isolate ions and to fragment them using collisionally activated decomposition (CAD) has been demonstrated using mixtures diluted to the low-femtomole level on target. Additionally, multiple stage mass spectrometry (MS/MS/MS) provides a second-generation product ion spectrum in which new fragment ions are detected and new stretches of amino acids are identified.     

Structural characterizations of lipids A by MS/MS of doubly charged ions on a hybrid linear ion trap/orbitrap mass spectrometer

Here, a new 'one pot' and fast approach is described, based on electrospray ionization (ESI) of negative ions by using a hybrid linear ion trap/orbitrap mass spectrometer (LTQ/orbitrap) for MS and MS/MS analysis. By this method the distribution of the primary and secondary acyl residues of the intact lipid A is inferred by analysis of the ESI spectra measured in positive and negative mode. The analysis of these data allows an unequivocal assignment of the fatty acid distribution. This methodology was successfully tested on two different lipid A with known structures, deriving from the Agrobacterium tumefaciens and Escherichia coli lipopolysaccharides (LPS).     

Rapid protein identification using a microscale electrospray LC/MS system on an ion trap mass spectrometer

A methodology has been developed for the rapid identification of gel separated proteins. Following in gel protein digestion with trypsin, the resulting peptide mixture is analyzed by on-line liquid chromatography, electrospray mass spectrometry (LC/MS). The mass spectral data containing either accurate mass values or sequence specific fragment ion information is then matched to a database of known protein sequences. Key features of the LC/MS system are the use of a novel integrated, microscale LC column-electrospray interface and variable flow solvent delivery to optimize the efficiency of sample loading and gradient elution. With these enhancements, only 10 min is required to analyze each sample. The method is routine for sample amounts ranging from 50 to 500 fmol. The analysis parameters for the ion trap mass spectrometer have to be carefully adjusted in order to keep pace with the rapidly eluting LC peaks. Although designed for rapid LC separations, the integrated column-electrospray interface is also able to provide extended analyses of selected components using a technique known as “peak parking. ”     

A two-dimensional quadrupole ion trap mass spectrometer

The use of a linear or two-dimensional (2-D) quadrupole ion trap as a high performance mass spectrometer is demonstrated. Mass analysis is performed by ejecting ions out a slot in one of the rods using the mass selective instability mode of operation. Resonance ejection and excitation are utilized to enhance mass analysis and to allow isolation and activation of ions for MS(n) capability. Improved trapping efficiency and increased ion capacity are observed relative to a three-dimensional (3-D) ion trap with similar mass range. Mass resolution comparable to 3-D traps is readily achieved, including high resolution at slower scan rates, although adequate mechanical tolerance of the trap structure is a requirement. Additional advantages of 2-D over 3-D ion traps are also discussed and demonstrated.     

Peptide photodissociation at 157 nm in a linear ion trap mass spectrometer

The photodissociation by 157 nm light of singly- and doubly-charged peptide ions containing C- or N-terminal arginine residues was studied in a linear ion trap mass spectrometer. Singly-charged peptides yielded primarily x- and a-type ions, depending on the location of the arginine residue, along with some related side-chain fragments. These results are consistent with our previous work using a tandem time-of-flight (TOF) instrument with a vacuum matrix-assisted laser desorption/ionization (MALDI) source. Thus, the different internal energies of precursor ions in the two experiments seem to have little effect on their photofragmentation. For doubly-charged peptides, the dominant fragments observed in both photodissociation and collisionally induced dissociation (CID) experiments are b- and y-type ions. Preliminary experiments demonstrating fragmentation of multiply-charged ubiquitin ions by 157 nm photodissociation are also presented.     

Dual linear ion trap/orthogonal acceleration time-of-flight mass spectrometer with improved precursor ion selectivity

A new hybrid mass spectrometer based on dual linear ion traps (LITs) and an orthogonal acceleration time-of-flight mass spectrometer (oaTOF), that can achieve MS(n) analysis and high-mass-accuracy detection with high sensitivity, has been developed. Dual-LIT was necessary because, in a single LIT plus oaTOF combination, the LIT pressure favorable for high precursor selectivity in MS(n) analysis (less than 1 mTorr) was far different from an optimum damping pressure (50-100 mTorr) for efficient connection to the TOF mass spectrometer. A dual-LIT solved this problem of inconsistency of the optimum pressures by using the first LIT for MS(n) analysis and the second LIT for collisional damping. This dual-LIT/TOF instrument achieved high-sensitivity MS(n) analysis with high precursor-ion selectivity.     

Rapid screening and characterization of drug metabolites using multiple ion monitoring dependent product ion scan and postacquisition data mining on a hybrid triple quadrupole‐linear ion trap mass spectrometer

Multiple ion monitoring (MIM)‐dependent acquisition with a triple quadrupole‐linear ion trap mass spectrometer (Q‐trap) was previously developed for drug metabolite profiling. In the analysis, multiple predicted metabolite ions are monitored in both Q1 and Q3 regardless of their fragmentations. The collision energy in Q2 is set to a low value to minimize fragmentation. Once an expected metabolite is detected by MIM, enhanced product ion (EPI) spectral acquisition of the metabolite is triggered. To analyze in vitro metabolites, MIM‐EPI retains the sensitivity and selectivity similar to that of multiple reaction monitoring (MRM)‐EPI in the analysis of in vitro metabolites. Here we present an improved approach utilizing MIM‐EPI for data acquisition and multiple data mining techniques for detection of metabolite ions and recovery of their MS/MS spectra. The postacquisition data processing tools included extracted ion chromatographic analysis, product ion filtering and neutral loss filtering. The effectiveness of this approach was evaluated by analyzing oxidative metabolites of indinavir and glutathione (GSH) conjugates of clozapine and 4‐ethylphenol in liver microsome incubations. Results showed that the MIM‐EPI‐based data mining approach allowed for comprehensive detection of metabolites based on predicted protonated molecules, product ions or neutral losses without predetermination of the parent drug MS/MS spectra. Additionally, it enabled metabolite detection and MS/MS acquisition in a single injection. This approach is potentially useful in high‐throughout screening of metabolic soft spots and reactive metabolites at the drug discovery stage. Copyright © 2009 John Wiley & Sons, Ltd.     

Protein identification via surface-induced dissociation in an FT-ICR mass spectrometer and a patchwork sequencing approach

Surface-induced dissociation (SID) and collision-induced dissociation (CID) are ion activation techniques based on energetic collisions with a surface or gas molecule, respectively. One noticeable difference between CID and SID is that SID does not require a collision gas for ion activation and is, therefore, directly compatible with the high vacuum requirement of Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers. Eliminating the introduction of collision gas into the ICR cell for collisional activation dramatically shortens the acquisition time for MS/MS experiments, suggesting that SID could be utilized for high-throughput MS/MS studies in FT-ICR MS. We demonstrate for the first time the utility of SID combined with FT-ICR MS for protein identification. Tryptic digests of standard proteins were analyzed using a hybrid 6-tesla FT-ICR mass spectrometer with SID and CID capabilities. SID spectra of mass-selected singly and doubly charged peptides were obtained using a diamond-coated target mounted at the rear trapping plate of the ICR cell. The broad internal energy distribution deposited into the precursor ion following collision with the diamond surface allowed a variety of fragmentation channels to be accessed by SID. Composition and sequence qualifiers produced by SID of tryptic peptides were used to improve the statistical significance of database searches. Protein identification MASCOT scores obtained using SID were comparable or better than scores obtained using sustained off-resonance irradiation collision-induced dissociation (SORI-CID), the conventional ion activation technique in FT-ICR MS.     

'N-in-one' strategy for metabolite identification using a liquid chromatography/hybrid triple quadrupole linear ion trap instrument using multiple dependent product ion scans triggered with full mass scan

This paper describes a new strategy that utilizes the fast trap mode scan of the hybrid triple quadrupole linear ion trap (QqQ(LIT)) for the identification of drug metabolites. The strategy uses information-dependent acquisition (IDA) where the enhanced mass scan (EMS), the trap mode full scan, was used as the survey scan to trigger multiple dependent enhanced product ion scans (EPI), the trap mode product ion scans. The single data file collected with this approach not only includes full scan data (the survey), but also product ion spectra rich in structural information. By extracting characteristic product ions from the dependent EPI chromatograms, we can provide nearly complete information for in vitro metabolites that otherwise would have to be obtained by multiple precursor ion scan (prec) and constant neutral loss (NL) analysis. This approach effectively overcomes the disadvantages of traditional prec and NL scans, namely the slow quadrupole scan speed, and possible mass shift. Using nefazodone (NEF) as the model compound, we demonstrated the effectiveness of this strategy by identifying 22 phase I metabolites in a single liquid chromatography/tandem mass spectrometry (LC/MS/MS) run. In addition to the metabolites reported previously in the literature, seven new metabolites were identified and their chemical structures are proposed. The oxidative dechlorination biotransformation was also discovered which was not reported in previous literature for NEF. The strategy was further evaluated and worked well for the fast discovery setting when a ballistic gradient elution was used, as well as for a simulated in vivo setting when the incubated sample (phase I metabolites) was spiked to control human plasma extract and control human urine.     

Rapid hydrocarbon analysis using a miniature rectilinear ion trap mass spectrometer

A miniature ion trap mass analyzer was applied to the analysis of traces of hydrocarbons and simple heteroatomics in the vapor phase and in aqueous solution. Vapors of acetone, acetic acid, acetonitrile, benzene, butanethiol, carbon disulfide, hexane, dichloromethane, naphthalene, toluene and xylenes were detected and quantified using solid sorbent trapping and, in some cases, by passage through a membrane interface. Aqueous solutions of benzene, toluene, xylenes, hexane and a petroleum naphtha distillate were examined using the membrane interface. Sampling, detection and identification of all compounds was completed in times of less than one minute. The gas-phase samples of toluene and benzene were detected at 200 ppt (limit of detection, LOD) for toluene and 600 ppt for benzene. Identification of benzene and xylene in aqueous solutions was readily achieved with LODs of 200 and 400 ppb, respectively. Quantification over a linear dynamic range of two orders of magnitude for the aqueous samples and three orders of magnitude for the vapor-phase samples was demonstrated.