An external secondary ion source for fourier transform mass spectrometry

A differentially pumped external secondary ion source for Fourier transform mass spectrometry is described. Installation does not interfere with other experiments such as laser desorption or photodissociation. Spectra of cesium iodide clusters (with ions up to m/z 8187), polyethylene glycol 1000, and histidine from both glycerol and dithiothreitol-dithioerythritol matrices are reported. The ion with m/z 156 from histidine was recorded with mass resolution of 160,000.     

High-resolution atmospheric pressure infrared laser desorption/ionization mass spectrometry imaging of biological tissue

An atmospheric pressure laser desorption/ionization mass spectrometry imaging ion source has been developed that combines high spatial resolution and high mass resolution for the in situ analysis of biological tissue. The system is based on an infrared laser system working at 2.94 to 3.10 μm wavelength, employing a Nd:YAG laser-pumped optical parametrical oscillator. A Raman-shifted Nd:YAG laser system was also tested as an alternative irradiation source. A dedicated optical setup was used to focus the laser beam, coaxially with the ion optical axis and normal to the sample surface, to a spot size of 30 μm in diameter. No additional matrix was needed for laser desorption/ionization. A cooling stage was developed to reduce evaporation of physiological cell water. Ions were formed under atmospheric pressure and transferred by an extended heated capillary into the atmospheric pressure inlet of an orbital trapping mass spectrometer. Various phospholipid compounds were detected, identified, and imaged at a pixel resolution of up to 25 μm from mouse brain tissue sections. Mass accuracies of better than 2 ppm and a mass resolution of 30,000 at m/z?=?400 were achieved for these measurements.

A high performance low magnetic field internal electrospray ionization-fourier transform ion cyclotron resonance mass spectrometer

A new in-magnetic field electrospray ionization (ESI) and Fourier transform ion cyclotron resonance mass spectrometer has been constructed and evaluated. This system is characterized by the use of multiple concentric cryopanels to achieve ultrahigh vacuum in the ion cyclotron resonance cell region, a probe-mounted internal ESI source, and a novel in-field shutter. Initial experiments demonstrate high resolution mass measurement capability at a field strength of 1 T. Mass resolution of 700,000 has been obtained for the 3+ charge state of Met-Lys-bradykinin (at m/z 440) generated by electrospray ionization. When electron impact ionization was employed, resolution in excess of 9,200,000 was achieved for nitrogen molecular ions (N ₂ ⁺ ). Isotopic resolution for molecular ions of bovine ubiquitin (MW=8565 µ) also was achieved by using small ion populations.     

Calibration Point for Electron Ionization MS / MS spectra measured with multiquadrupole mass spectrometers

A protocol for establishing standard instrument conditions for measurement of product ion MS/MS spectra from parent ions produced by electron ionization is presented. Within this protocol, the ion at m/z 231 (C₅F₉ ⁺) from perfluorokerosene or perfluorotributylamine is selected as the parent ion and subjected to collision-induced dissociation. The relative intensities of product ions at m/z 69, 131, and 181 are monitored as a function of collision energy while keeping the target gas pressure constant within the range of 10^?4–10^?6 torr (measured), or a beam attenuation of approximately 30-70%. The collision energy at which the ion intensities for product ions at m/z 69 and 181 are equal is defined as the calibration point at that collision gas pressure; the intensity of the ion at m/z 131 is very close to this value as well. Electron ionization MS/MS spectra taken at the calibration point using two different multiquadrupole instruments show good reproducibility for several test compounds. The high degree of similarity may aid in the establishment of a MS/MS spectral library.     

Analysis of dammar resin with MALDI‐FT‐ICR‐MS and APCI‐FT‐ICR‐MS

Comprehensive analysis of high‐resolution mass spectra of aged natural dammar resin obtained with Fourier transform ion cyclotron resonance mass spectrometer (FT‐ICR‐MS) using matrix‐assisted laser desorption/ionization (MALDI) and atmospheric pressure chemical ionization (APCI) is presented. Dammar resin is one of the most important components of painting varnishes. Dammar resin is a terpenoid resin (dominated by triterpenoids) with intrinsically very complex composition. This complexity further increases with aging. Ten different solvents and two‐component solvent mixtures were tested for sample preparation. The most suitable solvent mixtures for the MALDI‐FT‐ICR‐MS analysis were dichloromethane‐acetone and dichloromethane‐ethanol. The obtained MALDI‐FTMS mass spectrum contains nine clusters of peaks in the m/z range of 420–2200, and the obtained APCI‐FTMS mass spectrum contains three clusters of peaks in the m/z range of 380–910. The peaks in the clusters correspond to the oxygenated derivatives of terpenoids differing by the number of C₁₅H₂₄ units. The clusters, in turn, are composed of subclusters differing by the number of oxygen atoms in the molecules. Thorough analysis and identification of the components (or groups of components) by their accurate m/z ratios was carried out, and molecular formulas (elemental compositions) of all major peaks in the MALDI‐FTMS and APCI‐FTMS spectra were identified (and groups of possible isomeric compounds were proposed). In the MALDI‐FTMS and APCI‐FTMS mass spectrum, besides the oxidized C₃₀, triterpenoids also peaks corresponding to C₂₉ and C₃₁ derivatives of triterpenoids (demethylated and methylated, correspondingly) were detected. MALDI and APCI are complementary ionization sources for the analysis of natural dammar resin. In the MALDI source, preferably polar (extensively oxidized) components of the resin are ionized (mostly as Na⁺ adducts), whereas in the APCI source, preferably nonpolar (hydrocarbon and slightly oxidized) compounds are ionized (by protonation). Either of the two ionization methods, when used alone, gives an incomplete picture of the dammar resin composition. Copyright © 2012 John Wiley & Sons, Ltd.     

Calibration function for the orbitrap FTMS accounting for the space charge effect

Ion storage in an electrostatic trap has been implemented with the introduction of the Orbitrap Fourier transform mass spectrometer (FTMS), which demonstrates performance similar to high-field ion cyclotron resonance MS. High mass spectral characteristics resulted in rapid acceptance of the Orbitrap FTMS for Life Sciences applications. The basics of Orbitrap operation are well documented; however, like in any ion trap MS technology, its performance is limited by interactions between the ion clouds. These interactions result in ion cloud couplings, systematic errors in measured masses, interference between ion clouds of different size yet with close m/z ratios, etc. In this work, we have characterized the space-charge effect on the measured frequency for the Orbitrap FTMS, looking for the possibility to achieve sub-ppm levels of mass measurement accuracy (MMA) for peptides in a wide range of total ion population. As a result of this characterization, we proposed an m/z calibration law for the Orbitrap FTMS that accounts for the total ion population present in the trap during a data acquisition event. Using this law, we were able to achieve a zero-space charge MMA limit of 80 ppb for the commercial Orbitrap FTMS system and sub-ppm level of MMA over a wide range of total ion populations with the automatic gain control values varying from 10 to 10⁷.     

Development of medium pressure laser ionization,MPLI. Description of the MPLI ion source

A novel pulsed valve/ion source combination capable of time-resolved sampling from atmospheric pressure has been developed for use with laser ionization time of flight mass spectrometry. The source allows ionization extremely close to the nozzle of the pulsed valve, enabling ultra-sensitive detection of a number of compounds, e.g., NO, at mixing ratios <1 pptV. Furthermore, at analyte mixing ratios in the ppbV range, the temporal resolution of the system is in the sub-second regime, allowing time-resolved monitoring of highly dynamic and complex mixtures, e.g., human breath or reacting chemical mixtures in atmospheric smog chamber experiments. Rotational temperatures of approximately 50 K have been observed for analytes seeded in the supersonic jet expansion at a distance of 1 mm downstream of the nozzle orifice. The refinement of the original ion source has drastically reduced the impact of reflected laser light and the resultant electron impact signals previously observed. The general applicability of this technique is demonstrated here by coupling the source to commercially available as well as home-built time-of-flight mass spectrometers. Finally, we discuss the MPLI technique in view of the very recently introduced atmospheric pressure laser ionization (APLI) as well as the traditional jet-REMPI approach.     

Salicylaldehyde azine cluster formation observed by cold‐spray ionization mass spectrometry

We installed a cold‐spray ionization (CSI) source on a mass spectrometer to investigate the self‐assembly behavior of an aggregation‐induced emission enhancement system. Using a CSI source and the three‐dimensional platform, a self‐assembly system of a salicylaldehyde azine (SAA) was studied in mixture solution. This method permitted the determination of the structural information of the solution state, which cannot be detected by conventional mass spectrometry. In addition to the [M+H]⁺ ion (M is the SAA molecule), many major ion clusters such as [2M+Na]⁺ at m/z 503, [3M+Na]⁺ at m/z 743, [4M+Na]⁺ at m/z 983 and higher order aggregates were observed in the CSI mass spectra. However, many fragment ions, with the exception of cluster ions, appeared with high abundance when the ESI ion source was used due to the desolvation chamber temperature, suggesting that some aggregation can be detected at low temperatures. To investigate the effect of solvent on the aggregation, the CSI‐mass spectrometry (MS) experiments of SAA in absolute ethanol solution and ethanol/water (good/poor solvent) mixture solution were conducted. The most abundant ion peak was protonated SAA (m/z 241) in absolute ethanol, but many cluster ions and some multiple charged ion peaks were observed after adding a small amount of water into the ethanol solution. The results showed good agreement with that inferred by the combinational analysis of scanning electron microscope and fluorescence microscopy, indicating that CSI‐MS is capable of providing self‐assembly information of labile molecules in the solution state. Copyright © 2013 John Wiley & Sons, Ltd.     

Micro-Electrospray: Zeptomole/attomole per microliter sensitivity for peptides

The micro-electrospray ionization source has been optimized for the specific analysis of neuropeptides such as neurotensin and methionine enkephalin. The source has the option of integrating nanoliter flow-rate desalting and preconcentration techniques into the micro-electrospray spray needle, eliminating post-column dead volumes. For neurotensin, the most sensitive neuropeptide analyzed thus far in this work, the injection of 10 μL of a solution containing 320 zeptomolesy/gmL gave an [M + 3H]⁺³ ion at m/z 558.4 with S/N of > 8∶1. The MS/MS analysis of this peptide for the fragment ion at m/z 578.9 gave a S/N > 20∶1 for a solution containing 32 attomoles/μL.     

LC–Tandem-MS Development and Validation for the Determination of Tegaserod in Beagle Dog Plasma and Its Application to a Pharmacokinetic Study

A sensitive and selective liquid chromatography-tandem mass spectrometry method for quantitative determination of tegaserod was developed and validated over the linearity range 1.0–200.0 ng mL^?1 with 0.5 mL of plasma using diphenhydramine as an internal standard. Liquid–liquid extraction using ethyl ether was used to extract the drug and the internal standard from plasma. The mass spectrometer was operated under the selected reaction monitoring mode using the atmospheric pressure chemical ionization technique. The mobile phase consisted of methanol–water–formic acid (80:20:1, v/v/v), at a flow rate of 0.6 mL min^?1. In the positive mode, tegaserod produced a protonated precursor ion at m/z 302 and a corresponding product ion at m/z 173. The internal standard produced a protonated precursor ion at m/z 256 and a corresponding product ion at m/z 167. The intra- and inter-day accuracy at all levels fell in the ranges of 100.72–102.75% and 100.61–105.45%, and the intra- and inter-day precision were in the ranges of 4.20–5.74% and 1.90–4.17%, respectively. The method was successfully applied to a pharmacokinetic study of tegaserod after an oral administration of two kinds of tegaserod preparations to beagle dogs.     

Considerations for design of a Fourier transform mass spectrometer in the 4.2 K cold bore of a superconducting magnet

An external source Fourier transform mass spectrometer (FTMS) constructed inside the vertical cold bore of a superconducting magnet will have dramatic advantages in effective magnetic field, noise figures, and base pressure over current commercially available external source FTMS systems. There are substantial, but solvable, difficulties in the design, primarily with regard to control of the helium boiloff rate to an acceptable level, as well as relatively minor design challenges with heat sinks, contraction of metallic ion optic elements in the extreme temperature, and tandem mass spectrometry experiments. However, the ability to construct the FTMS inside the narrow bore tube of existing, commercially available vertical bore NMR magnets will allow access to the upper magnetic field limit currently used by 900 MHz (21 Tesla) - 1 GHz (23.3 Tesla) NMR experiments. The vacuum system, simply by being held inside the cold bore at 4.2 K, will cryopump itself dropping base pressures substantially, and heat sinking the input resistor of the preamplifier to this cryogenically cooled vacuum chamber will allow reduction of the input Johnson noise by a factor of 8.4 with associated 8.4-fold improvement in signal/noise, sensitivity, and dynamic range. The simultaneous improvement of three fundamental limiting factors in the FTMS (field strength, base pressure, and Johnson noise figure) will clearly outweigh the concomitant increased helium boiloff rate particularly if this rate can be dropped to the estimated <5 L/day range. The additional use of modern cryorefrigerators will further reduce helium boiloff to zero except during MS(n) experiments and system cooldown.     

Effect of ion-molecule collisions in the vacuum chamber of an electrospray time-of-flight mass spectrometer on mass spectra of proteins

Electrospray ionization spectra of positive multiply charged ions of several proteins with molecular weight from 8565 to 339,100 u were recorded at different pressures of residual gas in the vacuum chamber of an electrospray time-of-flight mass spectrometer. The pressure was varied in the range (0.2 to 5) × 10^?6 torr. The effect of pressure was found to be significant even for the lightest protein investigated (ubiquitin), which resulted in a decrease of both sensitivity and resolution. Investigations of the arrival-time distributions and the energy distributions of ions showed that collision-induced dissociation (CID) of the protein ions in the drift region is the main process responsible for the effect. Several CID cross sections of proteins were estimated from a series of mass spectra recorded at different pressures in the reflecting mode: 1150 Ų for cytochrome c (averaged over charge states z = 14–18), 800 Ų for lysozyme (z = 8–10), 1840 Ų for apomyoglobin (z = 12–25), 800 Ų for holomyoglobin (z = 8), and 2500 Ų for carbonic anhydrase II (z = 22–35). Several experiments with large proteins in their native conformations and low charge states (m/z 0,000) demonstrate that these ions are less sensitive to high residual gas pressure.     

Laser desorption VUV postionization MS imaging of a cocultured biofilm

Laser desorption postionization mass spectrometry (LDPI-MS) imaging is demonstrated with a 10.5 eV photon energy source for analysis and imaging of small endogenous molecules within intact biofilms. Biofilm consortia comprised of a synthetic Escherichia coli K12 coculture engineered for syntrophic metabolite exchange are grown on membranes and then used to test LDPI-MS analysis and imaging. Both E. coli strains displayed many similar peaks in LDPI-MS up to m/z 650, although some observed differences in peak intensities were consistent with the appearance of byproducts preferentially expressed by one strain. The relatively low mass resolution and accuracy of this specific LDPI-MS instrument prevented definitive assignment of species to peaks, but strategies are discussed to overcome this shortcoming. The results are also discussed in terms of desorption and ionization issues related to the use of 10.5 eV single-photon ionization, with control experiments providing additional mechanistic information. Finally, 10.5 eV LDPI-MS was able to collect ion images from intact, electrically insulating biofilms at ～100 μm spatial resolution. Spatial resolution of ～20 μm was possible, although a relatively long acquisition time resulted from the 10 Hz repetition rate of the single-photon ionization source.

Detection of Radical Adducts with Small Molecular Weights by Matrix-Assisted Laser Desorption/Ionization with Fourier Transform Mass Spectrometry

As an alternative method, matrix-assisted laser desorption/ionization with Fourier transform mass spectrometry （MALDI-FTMS） has been successfully used to detect and identify free radical adducts with small molecular weights of hydroxyl and 2-cyano-2-propyl radicals trapped with 5,5-dimethylpyrroline N-oxide （DMPO）. The detection and identification by MS/MS experiments using sustained offresonance irradiation collision-induced dissociation （SORI-CID） of [（DMPO＋·OH-·H）＋H^＋] （m/z 130.0868） and [DMPO＋2 ·CH（CH3）2CN＋H^＋] （m/z 250.1917） have demonstrated that MALDI-FTMS could be an effective method for detection and identification of free radical adducts. Other radical adducts have been also detected and identified. The approach of MALDI-FTMS is simple, fast, and sensitive which has potential for high-throughput analysis.     

High-resolution excitation of ions in a low-pressure linear ion trap

An exploration of the parameters necessary to obtain high‐resolution excitation, using dipolar excitation, of an ion in a linear ion trap has been undertaken in this study. These parameters included ion trap pressure, excitation amplitude, excitation period, drive frequency of the ion trap, Mathieu q value and the mass of the ion of interest. An understanding of how these parameters play a role in high‐resolution excitation is necessary to the development of a method for the targeted tandem mass spectrometric (MS/MS) analysis of ions with the same nominal mass. Resonance excitation profiles with full width half maxima as narrow as 0.015 m/z units could be obtained, under the right conditions, for an ion from a homogenously substituted triazatriphosphorine at m/z 322.049, which translates into a mass resolution of >21 500. In this particular case the requirement for high resolution was a low trap pressure (3.8 × 10^?5 Torr), low excitation amplitude (3 mV), long excitation period (100 ms) and a high Mathieu q value(0.8) when using a drive frequency of 1.228 MHz. Similar conditions were used to demonstrate the isolation of individual [M + H]⁺ component ions from mixtures of bromazepam (m/z 316.008)/chlorprothixene (m/z 316.0921)/fendiline (m/z 316.206) and chlorprothixene (m/z 316.0921)/oxycodone (m/z 316.1543)/fendiline (m/z 316.206) prior to obtaining product ion spectra with excitation at q = 0.236. In the former mixture the individual components were isolated with near 100% efficiency while in the latter mixture the isolation efficiency dropped to near 50% for the oxycodone component and to 80% for the other components. Copyright © 2010 John Wiley & Sons, Ltd.     

Ion trajectories in an electrostatic ion guide for external ion source fourier transform ion cyclotron resonance mass spectrometry

An electrostatic ion guide (EIG) that consists of concentric cylinder and central wire electrodes can transport ions efficiently from an external ion source to an ion cyclotron resonance (ICR) ion trap for mass analysis, with several advantages over current injection methods. Because the electrostatic force of the EIG captures ions in a stable orbit about the wire electrode, ions with initially divergent trajectories may be redirected toward the ICR ion trap for improved ion transmission efficiency. SIMION trajectory calculations (ion kinetic energy, 1–200 eV; elevation angle, 0.30 °; azimuthal angle, 0.360°) predict that ions of m/z 1000 may be transmitted through a strong (0.01 → 3.0-T) magnetic field gradient. Judicious choice of ion source position and EIG potential minimizes the spread in ion axial kinetic energy at the ICR ion trap. Advantages of the EIG include large acceptance angle, even for ions that have large initial kinetic energy and large radial displacement with respect to the central z-axis, low ion extraction voltage (5–20 V), and efficient trapping because ions need not be accelerated to high velocity to pass through the magnetic field gradient.     

High field asymmetric waveform ion mobility spectrometry-mass spectrometry: an investigation of leucine enkephalin ions produced by electrospray ionization

High field asymmetric waveform ion mobility spectrometry (FAIMS) provides atmospheric pressure, room temperature, low-resolution separation of gas-phase ions. The FAIMS analyzer acts as an ion filter that can continuously transmit one type of ion, independent of m/z. The combination of FAIMS with electrospray ionization and mass spectrometry (ESI-FAIMS-MS) is a powerful technique and is used in this study to investigate the cluster ions of leucine enkephalin (YGGFL). Separation by FAIMS of leucine enkephalin ions having the same m/z (m/z 556.5), [M + H]⁺ and [2M + 2H]²⁺, was observed. In addition, four complex ions of leucine enkephalin, [2M + H]⁺, [4M + 2H]²⁺, [6M + 3H]³⁺, and [8M + 4H]⁴⁺, all having m/z 1112, were shown to be separated in FAIMS. Fragmentation of ions as the result of harsh conditions within the mass spectrometer interface (FAIMS-MS) was shown to provide similar information to that obtained from MS/MS experiments in conventional ESI-MS.     

High-resolution accurate mass measurements of biomolecules using a new electrospray ionization ion cyclotron resonance mass spectrometer

A novel electrospray ionization/Fourier transform ion cyclotron resonance mass spectrometer based on a 7-T superconducting magnet was developed for high-resolution accurate mass measurements of large biomolecules. Ions formed at atmospheric pressure using electrospray ionization (ESI) were transmitted (through six differential pumping stages) to the trapped ion cell maintained below 10^?9 torr. The increased pumping speed attainable with cryopumping (> 10⁵ L/s) allowed brief pressure excursions to above 10^?4 torr, with greatly enhanced trapping efficiencies and subsequent short pumpdown times, facilitating high-resolution mass measurements. A set of electromechanical shutters were also used to minimize the effect of the directed molecular beam produced by the ES1 source and were open only during ion injection. Coupled with the use of the pulsed-valve gas inlet, the trapped ion cell was generally filled to the space charge limit within 100 ms. The use of 10–25 ms ion injection times allowed mass spectra to be obtained from 4 fmol of bovine insulin (M_r 5734) and ubiquitin (M_r 8565, with resolution sufficient to easily resolve the isotopic envelopes and determine the charge states. The microheterogeneity of the glycoprotein ribonuclease B was examined, giving a measured mass of 14,898.74 Da for the most abundant peak in the isotopic envelope of the normally glycosylated protein (i.e., with five mannose and two N-acetylglucosamine residues (an error of approximately 2 ppm) and an average error of approximately 1 ppm for the higher glycosylated and various H₃PO₄ adducted forms of the protein. Time-domain signals lasting in excess of 80 s were obtained for smaller proteins, producing, for example, a mass resolution of more than 700,000 for the 4⁺ charge state (m/z 1434) of insulin.     

Mechanism of Formation of the Major Estradiol Product Ions Following Collisional Activation of the Molecular Anion in a Tandem Quadrupole Mass Spectrometer

The importance of the mass spectral product ion structure is highlighted in quantitative assays, which typically use multiple reaction monitoring (MRM), and in the discovery of novel metabolites. Estradiol is an important sex steroid whose quantitation and metabolite identification using tandem mass spectrometry has been widely employed in numerous clinical studies. Negative electrospray ionization tandem mass spectrometry of estradiol (E2) results in several product ions, including the abundant m/z 183 and 169. Although m/z 183 is one of the most abundant product ions used in many quantitative assays, the structure of m/z 183 has not been rigorously examined. We suggest a structure for m/z 183 and a mechanism of formation consistent with collision induced dissociation (CID) of E2 and several stable isotopes ([D₄]-E2, [¹³C₆]-E2, and [D₁]-E2). An additional product ion from E2, namely m/z 169, has also been examined. MS³ experiments indicated that both m/z 183 and m/z 169 originate from only E2 [M – H]^– m/z 271. These ions, m/z 183 and m/z 169, were also present in the collision induced decomposition mass spectra of other prominent estrogens, estrone (E1) and estriol (E3), indicating that these two product ions could be used to elucidate the estrogenic origin of novel metabolites. We propose two fragmentation schemes to explain the CID data and suggest a structure of m/z 183 and m/z 169 consistent with several isotopic variants and high resolution mass spectrometric measurements.      

First signal on the cryogenic fourier-transform ion cyclotron resonance mass spectrometer

The construction and achievement of the first signal on a cryogenic Fourier-transform ion cyclotron resonance mass spectrometer (FTICR-MS) are reported here, demonstrating proof-of-concept of this new instrument design. Building the FTICR cell into the cold bore of a superconducting magnet provided advantages over conventional warm bore design. At 4.2 K, the vacuum system cryopumps itself, thus removing the requirement for a large bore to achieve the desired pumping speed for maintaining base pressure. Furthermore, because the bore diameter has been reduced, the amount of magnet wire needed to achieve high field and homogeneity was also reduced, greatly decreasing the cost/Tesla of the magnet. The current instrument implements an actively shielded 14-Tesla magnet of vertical design with an external matrix-assisted laser desorption/ionization (MALDI) source. The first signal was obtained by detecting the laser desorbed/ionized (LDI) C(60)(+*) ions, with the magnet at 7 Tesla, unshimmed, and the preamplifier mounted outside of the vacuum chamber at room temperature. A subsequent experiment done with the magnet at 14 Tesla and properly shimmed produced a C(60) spectrum showing approximately 350,000 resolving power at m/z approximately 720. Increased magnetic field strength improves many FTMS performance parameters simultaneously, particularly mass resolving power and accuracy.