Secondary ionization of chemical warfare agent simulants: atmospheric pressure ion mobility time-of-flight mass spectrometry

For the first time, the use of a traditional ionization source for ion mobility spectrometry (radioactive nickel ((63)Ni) beta emission ionization) and three alternative ionization sources (electrospray ionization (ESI), secondary electrospray ionization (SESI), and electrical discharge (corona) ionization (CI)) were employed with an atmospheric pressure ion mobility orthogonal reflector time-of-flight mass spectrometer (IM(tof)MS) to detect chemical warfare agent (CWA) simulants from both aqueous- and gas-phase samples. For liquid-phase samples, ESI was used as the sample introduction and ionization method. For the secondary ionization (SESI, CI, and traditional (63)Ni ionization) of vapor-phase samples, two modes of sample volatilization (heated capillary and thermal desorption chamber) were investigated. Simulant reference materials, which closely mimic the characteristic chemical structures of CWA as defined and described by Schedule 1, 2, or 3 of the Chemical Warfare Convention treaty verification, were used in this study. A mixture of four G/V-type nerve simulants (dimethyl methylphosphonate, pinacolyl methylphosphonate, diethyl phosphoramidate, and 2-(butylamino)ethanethiol) and one S-type vesicant simulant (2-chloroethyl ethyl sulfide) were found in each case (sample ionization and introduction methods) to be clearly resolved using the IM(tof)MS method. In many cases, reduced mobility constants (K(o)) were determined for the first time. Ion mobility drift times, flight times, relative signal intensities, and fragmentation product signatures for each of the CWA simulants are reported for each of the methods investigated.     

Electrospray ionization high-resolution ion mobility spectrometry for the detection of organic compounds, 1. Amino acids

Our aim in this investigation was to demonstrate the potential of the high-resolution electrospray ionization ion mobility spectrometry (ESI-IMS) technique as an analytical separation tool in analyzing biomolecular mixtures to pursue astrobiological objectives of searching for the chemical signatures of life during an in-situ exploration of solar system bodies. Because amino acids represent the basic building blocks of life, we used common amino acids to conduct the first part of our investigation, which is being reported here, to demonstrate the feasibility of using the ESI-IMS technique for detection of the chemical signatures of life. The ion mobilities of common amino acids were determined by electrospray ionization ion mobility spectrometry using three different drift gases (N2, Ar, and CO2). We demonstrated that the selectivity can be vastly improved in ion mobility spectroscopy (IMS) in detecting organic molecules by using different drift gases. When a judicial choice of drift gas is made, a vastly improved separation of two different amino acid ions resulted. It was found that each of the studied amino acids could be uniquely identified from the others, with the exception of alanine and glycine, which were never separable by more then 0.1 ms. This unique identification is a result of the different polarizabilities of the various drift gases. In addition, a better separation was achieved by changing the drift voltage in successive experimental runs without significantly degrading the resolution. We also report the result of our analysis of liquid samples containing mixtures of amino acids.     

Separation of cisplatin and its hydrolysis products using electrospray ionization high-field asymmetric waveform ion mobility spectrometry coupled with ion trap mass spectrometry

Cisplatin and its mono- and dihydrated complexes have been separated using a high-field asymmetric waveform ion mobility spectrometry (FAIMS) analyzer interfaced with electrospray ionization (ESI) and ion trap mass spectrometry (ITMS). The addition of helium to the nitrogen curtain/carrier gas in the FAIMS device improved both the sensitivity and selectivity of the electrospray analysis. Introduction of a three-component mixture as curtain/carrier gas, nitrogen, helium, and carbon dioxide, resulted in further improvements to sensitivity. Compared with conventional ESI-MS, the background chemical noise in the ESI-FAIMS-ITMS spectrum was dramatically reduced, resulting in over 30-fold improvement in the signal-to-noise ratio for cisplatin. Analytical results were linear over the concentration range 10-200 ng/mL for intact cisplatin with a corresponding detection limit determined of 0.7 ng/mL with no derivatization or chromatographic separation prior to analysis.     

Quantitation of amphetamine,methamphetamine, and their methylenedioxy derivatives in urine by solid-phase microextraction coupled with electrospray ionization-high-field asymmetric waveform ion mobility spectrometry-mass spectrometry

Amphetamine, methamphetamine, and their methylenedioxy derivatives have been identified and measured in a human urine matrix using solid-phase microextraction (SPME) and high-field asymmetric waveform ion mobility spectrometry (FAIMS) in combination with electrospray ionization (ESI) and mass spectrometric detection (MS). Limits of detection in human urine between 200 pg/mL and 7.5 ng/mL have been achieved. The use of a simple extraction method, SPME, combined with the high sensitivity and selectivity of ESI-FAIMS-MS eliminates the need for chromatographic separation and allows for very rapid sample processing.     

Ultrasensitive identification of localization variants of modified peptides using ion mobility spectrometry

Localization of the modification sites on peptides is challenging, particularly when multiple modifications or mixtures of localization isomers (variants) are involved. Such variants commonly coelute in liquid chromatography and may be undistinguishable in tandem mass spectrometry (MS/MS) for lack of unique fragments. Here, we have resolved the variants of singly and doubly phosphorylated peptides employing drift tube ion mobility spectrometry (IMS) coupled to time-of-flight mass spectrometry. Even with a moderate IMS resolving power of ～80-100, substantial separation was achieved for both 2+ and 3+ ions normally generated by electrospray ionization, including for the variants indistinguishable by MS/MS. Variants often exhibit a distribution of 3-D conformers, which can be adjusted for optimum IMS separation by prior field heating of ions in a funnel trap. The peak assignments were confirmed using MS/MS after IMS separation, but known species could be identified using just the ion mobility "tag". Avoiding the MS/MS step lowers the detection limit of localization variants to <100 amol, an order of magnitude better than that provided by electron transfer dissociation in an Orbitrap MS.     

Gas-phase proton-transfer chemistry coupled with TOF mass spectrometry and ion mobility-MS for the facile analysis of poly(ethylene glycols) and PEGylated polypeptide conjugates

Gas-phase ion/molecule chemistry has been combined with ion mobility separation and time-of-flight mass spectrometry to enable the characterization of large poly(ethylene glycol)s (PEGs) and PEGylated molecules (>40 kDa). A facile method is presented in which gas-phase superbases are reacted in the high-pressure source region of commercial TOF mass spectrometers to manipulate the charge states of large ions generated by electrospray ionization (ESI). Charge stripping decreases the spectral congestion typically observed in ESI mass spectra of high molecular weight polydisperse PEGylated molecules. From these data, accurate average molecular weights and molecular weight distributions for synthetic polymers and PEGylated proteins are determined. The average MW measured for PEGylated Granulocyte colony-stimulating factor (rh-GCSF, 40 726.2 Da) is in good agreement with the theoretical value, and a 16 Da mass shift is easily observed in the spectrum of an oxidized form of the heterogeneous PEGylated protein. Ion mobility separations can fractionate PEGs of different chain length; when coupled with charge stripping ion/molecule reactions, ion mobility mass spectrometry (IMMS) offers several analytical advantages over mass spectrometry alone for the characterization of large PEGylated molecules including enhanced dynamic range, increased sensitivity, and specificity. Low abundance free PEG in a PEGylated peptide preparation, which is not directly detectable by mass spectrometry, can be easily observed and accurately quantified with gas-phase ion/molecule chemistry combined with ion mobility mass spectrometry.     

Enhanced direct ambient analysis by differential mobility-filtered desorption electrospray ionization-mass spectrometry

Desorption electrospray ionization (DESI) is rapidly becoming established as one of the most powerful ionization techniques allowing direct surface analysis by mass spectrometry (MS) in the ambient environment. DESI provides a significant number of unique analytical capabilities for a broad range of applications, both quantitative and qualitative in nature including biological tissue imaging, pharmaceutical quality control, in vivo analysis, proteomics, metabolomics, forensics, and explosives detection. Despite its growing adoption as a powerful high throughput analysis tool, DESI-MS analysis at trace levels often suffers from background chemical interferences generated during the electrospray ionization processes. In order to improve sensitivity and selectivity, a differential mobility (DM) ion separation cell was successfully interfaced to a custom-built DESI ion source. This new hybrid platform can be operated in two modes: the "DM-off" mode for standard DESI analysis and "DM-on mode" where DESI-generated ions are detected after discrimination by the differential mobility cell. The performance of the DESI-DM-MS platform was tested with several samples typically amenable to DESI analysis, including counterfeit pharmaceuticals and binary mixtures of isobaric chemicals of importance in the pharmaceutical and food industries. In the DM-on mode, DESI-MS signal-to-noise ratios were improved by 70-190% when compared to the DM-off mode. Also, the addition of the DM cell enabled selective in-source ion activation of specific DESI-generated precursor ions, providing tandem MS-like spectra in a single stage mass spectrometer.     

Frequency dependence of alternating current electrospray ionization mass spectrometry

The novel effects resulting from the entrainment of low mobility ions during alternating current (ac) electrospray ionization are examined through mass spectrometry and voltage/current measurements. Curious phenomena such as pH modulation at high frequencies (>150 kHz) of an applied ac electric field are revealed and explained using simple mechanistic arguments. Current measurements are utilized to supplement these observations, and a simplified one-dimensional transient diffusion model for charge transport is used to arrive at a scaling law that provides better insight into the ac electrospray ionization process. Moreover, because of the different pathway for ion formation in comparison to direct current (dc) electrospray, ac electrospray (at frequencies >250 kHz) is shown to reduce the effects of ionization suppression in a mixture of two molecules with different surface activities.     

Separation of isomeric peptides using electrospray ionization/high-resolution ion mobility spectrometry

In this paper, the first examples of baseline separation of isomeric macromolecules by electrospray ionization/ion mobility spectrometry (ESI/IMS) at atmospheric pressure are presented. The behavior of a number of different isomeric peptides in the IMS was investigated using nitrogen as a drift gas. The IMS was coupled to a quadrupole mass spectrometer, which was used for identification and selective detection of the electrosprayed ions. The mobility data were used to determine their average collision cross sections. The gas-phase ions of isomeric peptides were found to have different collision cross sections. In all cases, doubly charged ions exhibited significantly (8-20%) larger collision cross sections than the respective singly charged species. The analysis of mixtures of the isomeric peptides clearly demonstrated the capability of IMS to separate gas-phase peptide ions due to small differences in their conformational structures, which cannot be determined by mass spectrometry. An actual resolving power of 80 was achieved for two doubly charged reversed sequenced pentapeptides. Baseline separation was provided for ions differing by only 2.5% in their measured collision cross sections; partial separation was shown for isomeric ions exhibiting differences as small as 1.1%.     

Coronaspray nebulization and ionization of liquid samples for ion mobility spectrometry

Ion mobility spectrometry after electrospray nebulization and ionization was investigated as a method for the detection of components dissolved in liquids. While electrosprary operating conditions proved promising, greater sensitivity was achieved when the electric potential applied to the sample introduction needle was increased above breakdown potential and a corona discharge was established. Passing the liquid through the corona discharge established a "coronaspray" that efficiently nebulized and ionized the solvent and analytes. In this initial investigation of coronaspray ion mobility spectrometry (CIMS), ion current as a function of potential, temperature, and liquid flow rate was studied; several IMS spectra were obtained; and a continuous monitoring mode of operation was demonstrated. The results from this study indicated that CIMS has potential as a versatile and sensitive detection method for a variety of analytical procedures involving liquid flowing streams such as flow injection analysis, liquid chromatography, capillary zone electrophoresis, and field flow fractionation.     

Miniaturized ultra high field asymmetric waveform ion mobility spectrometry combined with mass spectrometry for peptide analysis

Miniaturized ultra high field asymmetric waveform ion mobility spectrometry (ultra-FAIMS) combined with mass spectrometry (MS) has been applied to the analysis of standard and tryptic peptides, derived from α-1-acid glycoprotein, using electrospray and nanoelectrospray ion sources. Singly and multiply charged peptide ions were separated in the gas phase using ultra-FAIMS and detected by ion trap and time-of-flight MS. The small compensation voltage (CV) window for the transmission of singly charged ions demonstrates the ability of ultra-FAIMS-MS to generate pseudo-peptide mass fingerprints that may be used to simplify spectra and identify proteins by database searching. Multiply charged ions required a higher CV for transmission, and ions with different amino acid sequences may be separated on the basis of their differential ion mobility. A partial separation of conformers was also observed for the doubly charged ion of bradykinin. Selection on the basis of charge state and differential mobility prior to tandem mass spectrometry facilitates peptide and protein identification by allowing precursor ions to be identified with greater selectivity, thus reducing spectral complexity and enhancing MS detection.     

Design for electrospray ionization-ion mobility spectrometry

In this study, a new design for electrospray ionization ion mobility spectrometry (ESI-IMS) was developed. This design has two important differences in comparison to the present ESI-IMS systems. First, a few centimeters of the cell comprising the electrospray needle was located outside of the oven used for heating the IMS cell. This modification prevents prespray solvent evaporation problems such as needle clogging and disturbance of the electrospray process. Second, in addition to the drift gas, a counterflow of a heated gas (desolvation gas) was used between the counter electrode and the ion gate to speed up the desolvation process (Hill, H. H., Jr. Anal. Chem. 1998, 70, 4929-4938). This modification increased the solvent evaporation and resulted in decreasing the drift time, increasing the peak intensity and increasing the resolving power (RP) or enhancing the resolution for separation of two adjacent ion peaks. In this work, the ion mobility spectra of different compounds including ethion, malathion, metalaxyl, fenamifos, methylamine, triethylamine, tributhylamine, codeine, and morphine were obtained to confirm enhancing of the resolving power of the ion peaks by using the desolvation gas. Furthermore, the method has also been applied to obtain the figures of merit for ethion as a test compound. The linear dynamic range for ethion was in the range 50-1000 microg/L with a limit of quantification of the 50 microg/L.     

Characterization of phosphoantigens by high-performance anion-exchange chromatography-electrospray ionization ion trap mass spectrometry and nanoelectrospray ionization ion trap mass spectrometry

New phosphorylated microbial metabolites referred to as phosphoantigens activate immune responses in humans. Although these molecules have leading applications in medical research, no direct method allows their rapid and unambiguous structural identification. Here, we interfaced online HPAEC (high performance anion-exchange chromatography) with ESI-ITMS (electrospray ionization ion trap mass spectrometry) to identify such pyrophosphorylated molecules. A self-regenerating anion suppressor located upstream of electrospray ionization enabled the simultaneous detection of pyrophosphoester by conductimetry, UV and MS. By HPAEC-ITMS and HPAEC-ITMS2, a single run permitted characterization of reference phosphoantigens and of related structures. Although all compounds were resolved by HPAEC, MS enabled their detection and identification by [M-H]- and fragment ions. Isobaric phosphoantigen analogues were also separated by HPAEC and distinguished by MS2. The relevance of this device was demonstrated for phosphoantigens analysis in human urine and plasma. Furthermore, identification of natural phosphoantigens by automatically generated 2D mass spectra from nano-ESI-ITMS is presented. This last technique permits the simultaneous performance of molecular screening of natural phosphoantigen extracts and their identification.     

Simple coupling of gas chromatography to electrospray ionization mass spectrometry

A simple method for direct coupling of gas chromatography (GC) with electrospray ionization mass spectrometry (ESI/MS) has been developed. The outlet of the GC capillary column was placed between the ESI needle and the atmospheric pressure ionization (API) source of a mass spectrometer. The ionization occurs via dissolution of neutral compounds into the charged ESI droplet followed by ion evaporation or via a gas-phase proton transfer reaction between a protonated solvent molecule and an analyte. The mass spectra of organic volatile compounds showed abundant protonated molecules with little fragmentation, being very similar to those produced by normal liquid ESI. The quantitative performance of the system was evaluated by determining the limit of detection (LOD), linearity ( r (2)), and repeatability (RSD). The GC-ESI/MS method was shown to be stable, providing high sensitivity and good quantitative performance.     

Mass analysis of mobility-selected ion populations using dual gate, ion mobility, quadrupole ion trap mass spectrometry

An electrospray ionization, dual gate, ion mobility, quadrupole ion trap mass spectrometer (ESI-DG-IM-QIT-MS) was constructed and evaluated for its ability to select mobility-filtered ions prior to mass analysis. While modification of the common signal-averaged ion mobility experiment was required, no modifications to the QIT were necessary. The dual gate scanning mode of operation was used to acquire mobility spectra, whereas the single mobility monitoring experiment selectively filtered ions for concentration and subsequent fragmentation within the QIT. Ion mobility separation of positively charged peptides and negatively charged carbohydrates, followed by MS fragmentation, was demonstrated. For a 1-min acquisition time, it was possible to obtain complete de novo sequence information for the examined peptides. Fragmentation of the negative carbohydrate chlorine adducts yielded ions characteristic of cross-ring and glycosidic bond cleavage. Previous unions of atmospheric pressure ion mobility and mass spectrometry have been limited in their ability to reproducibly obtain MSn data for mobility separation ions. The union of high-pressure ion mobility with quadrupole ion trap mass spectrometry presents the unique opportunity to obtain more detailed information regarding the chemistries of gas-phase ions.     

Identification of weak and strong organic acids in atmospheric aerosols by capillary electrophoresis/mass spectrometry and ultra-high-resolution fourier transform ion cyclotron resonance mass spectrometry

A novel approach using a combination of capillary electrophoresis/mass spectrometry (CE/MS) and off-line Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) revealed the structural details of acidic constituents of atmospheric organic aerosol. Both techniques utilized electrospray ionization (ESI), a soft ionization method, to facilitate the analysis of complex mixtures of organic compounds. CE/ESI-MS using an UltraTrol LN-precoated capillary and acidic background electrolytes at different pH values (2.5 and 4.7) was used to differentiate between weak (carboxylic) and strong (sulfonic) organic acids. On the basis of the electrophoretic mobility, m/z constraints from CE/ESI(-)-MS, and elemental composition information retrieved from off-line FTICR-MS, a variety of aliphatic and aromatic carboxylic acids (CHO-bearing molecules), nitrogen-containing carboxylic acids (CHON-bearing molecules), organosulfates (CHOS-bearing molecules), and (nitrooxy)organosulfates (CHONS-bearing molecules) were tentatively identified in the Oasis-HLB-extracted urban PM(2.5) (particulate matter with an aerodynamic diameter of <2.5 μm). The chemical known/unknown structures of detected compounds were confirmed by the semiempirical Offord model (effective mobility linearly correlated to Z/M(2/3)). The majorities of the identified compounds are products of atmospheric reactions and are known contributors to secondary organic aerosols.     

Enantiomer separation of amino acids by complexation with chiral reference compounds and high-field asymmetric waveform ion mobility spectrometry: preliminary results and possible limitations

We present a new method for separation of enantiomers with high-field asymmetric waveform ion mobility spectrometry (FAIMS), coupled to mass spectrometric detection. Upon addition of an appropriate chiral reference compound to the analyte solution and subsequent ionization of the solution by electrospray ionization, analyte enantiomers formed diastereomeric complexes, which were potentially separable by FAIMS. The methodology being developed is intended to be general, but here amino acid analytes are specifically considered. In the examples presented herein, six pairs of amino acid enantiomers were successfully separated as metal-bound trimeric complexes of the form [MII(L-Ref)2(D/L-A)-H]+, where MII is a divalent metal ion, L-Ref is an amino acid in its L form acting as chiral reference compound, and A is the amino acid analyte. For example, D- and L-tryptophan were separated in FAIMS as [NiII(L-Asn)2(D-Trp)-H]+ and [NiII(L-Asn)2(L-Trp)-H]+. As FAIMS separation typically takes place over a time scale of only a few hundred milliseconds, the presented separation method opens new possibilities for rapid analysis of one analyte enantiomer in the presence of the other enantiomer. Preliminary quantification results are presented, which suggest that fast and sensitive quantitative chiral analyses can be performed with FAIMS. Method limitations are discussed in terms of diverse phenomena, which are not yet understood.     

Surface ionization ion mobility spectrometry

A surface ionization (SI) source was designed and constructed for ion mobility spectrometry (IMS). Compared with a conventional (63)Ni source, the surface ionization source is as simple and reliable, has an extended dynamic response range, is more selective in response, and does not have regulatory problems associated with radioactive ionization sources. The performance of this SI-IMS was evaluated with several different classes of compounds. Triethylamine was employed for studying the behavior of the ionization source under different source conditions and gaseous environments. Amines, tobacco alkaloids, and triazine herbicides were also investigated. Picogram level detection limits were achieved for target compounds with a response dynamic range of 5 orders of magnitude. Selective monitoring by IMS was also demonstrated. While the surface ionization source does not have the universality of response that is obtained with a (63)Ni ionization source, it is an excellent nonradioactive alternative for the ionization and ion mobility detection of those compounds to which it responds.     

Nontarget analysis of urine by electrospray ionization-high field asymmetric waveform ion mobility-tandem mass spectrometry

Nearly a decade after first commercialization, high field asymmetric waveform ion mobility spectrometry (FAIMS) has yet to find its place in routine chemical analysis. Prototypes have been used to demonstrate the utility of this separation technique combined with mass spectrometry (MS). Unfortunately, first generation commercial FAIMS instruments have gone practically unused by early adopters. Here, we show this to be due to poor ion transmission in the FAIMS-MS source interface. We present simple instrumental modifications and optimization of experimental conditions to achieve good performance from the first generation commercial FAIMS device (the Ionalytics Selectra) coupled to a high resolution Q-TOF-MS. In combination with nanospray ionization, we demonstrate for the first time the nontarget analysis of urine by FAIMS with minimal sample preparation. We show the unique suitability of electrospray ionization (ESI)-FAIMS-MS for identification of low abundance species such as urinary biomarkers of damage of nucleic acids in a complex biological matrix. The elimination of electrospray noise and matrix components by FAIMS and the continuous flow of analytes through FAIMS for accurate and tandem mass analysis produce high quality spectral data suitable for structural identification of unknowns. These characteristics make ESI-FAIMS-MS ideal for nontarget identification, even when compared to high efficiency LC-ESI-MS.