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V-type nerve agents are among the most toxic organophosphorus chemical warfare agents, and they are under strict regulation and supervision by the OPCW (Organization for the Prohibition of Chemical Weapons). The V-type class of materials refers to a potentially large number of analogues and isomers. In order to expose instances of unfulfillment of the OPCW treaty, it is essential to have the ability to detect and identify "unknown" analogues of this family, even in the absence of an analytical standard. This work demonstrates a new automated tool for the detection and identification of V-type analogues, using high-resolution-accurate-mass LC-MS analysis, followed by "Compound Discoverer" software data processing. This software, originally developed for metabolism and metabolomics screening, is used here to automatically detect various V-type analogues by picking peaks and comparing them to "in-silico" calculated modifications made on a predefined basic V-backbone structure (according to the OPCW definitions for V-type agents). Subsequently, a complete structural elucidation for the proposed molecular formula is obtained by MS/MS data analysis of the suspected component, for both the V-type analogue (using ESI(+) analysis) as well as its hydrolysis product (using ESI(-) analysis) for a better elucidation of the phosphonate "head" structure. This method was found to be useful for the detection and identification of several "unknown" analogues, at low ng/mL levels in soil extracts.

Highly toxic organophosphorous nerve agents (OPAs) have been used in several armed conflicts and terror attacks in the last few decades. A new method for retrospective determination of alkyl methylphosphonic acid (AMPA) metabolites in urine after exposure to VX, GB and GF nerve agents was developed. This method enables a rapid, sensitive and selective determination of trace levels of the nerve agent biomarkers ethyl methylphosphonic acid (EMPA), isopropyl methylphosphonic acid (IMPA) and cyclohexyl methylphosphonic acid (CMPA) in urine. The new technique involves a unique combination of two solid phase extraction (SPE) cartridges: a Ba/Ag/H cartridge for urine interference removal, and a ZrO2 cartridge for selective reconstitution and enrichment of the AMPAs. Extraction of AMPAs from the ZrO2 cartridge was accomplished with a 1% ammonium hydroxide (NH4OH) solution and was followed by analysis via liquid chromatography–mass spectrometry (LC–MS). The limits of quantitation (LOQs) were in the range of 10–100 pg/mL with recoveries of 64–71% (± 5–19%) after fast sample preparation and a total LC–MS analysis cycle time of 15 min and 13 min, respectively. This method was successfully applied in vivo in a rabbit that was exposed to 0.5 LD50 (7.5 µg/kg, i.v.) sarin for retrospective monitoring of the IMPA metabolite in urine. For the first time, IMPA was determined in rabbit urine samples for 15 days post-exposure, which is longer than any reported post-exposure method for AMPAs. To the best of our knowledge, this new method is the most sensitive and rapid for AMPA determination in urine by LC–MS/MS analysis.

The highly toxic nerve agent sarin (o-isopropyl methyl-phosphonofluoridate, GB) has been used in several armed conflicts and terror attacks in recent decades. Due to its inherent high sensitivity, liquid chromatography–mass spectrometry (LC–MS/MS) has the potential to detect ultratrace levels of fluoride-regenerated G and V agents after appropriate chemical derivatization. A new method for the retrospective determination of exposure to sarin was developed. The method is based on sarin regeneration from blood using the fluoride-induced technique followed by derivatization with 2-[(dimethylamino)methyl]phenol (2-DMAMP) and LC–ESI–MS/MS (MRM) analysis. The validated method presents good linear response in the concentration range of 5–1000 pg/mL with a limit of quantitation (LOQ) of 5 pg/mL, 13.8% accuracy, 16.7% precision and a total recovery of 62% ± 9%. This new analytical approach has several advantages over existing GC/GC–MS-based methods in terms of sensitivity, specificity and simplicity, in addition to a short LC–MS cycle time of 12 min. The method was successfully applied in an in vivo experiment for retrospective determination of sarin in a rabbit exposed to 0.1 LD50 sarin (1.5 µg/kg, i.v.). GB-2-DMAMP was easily determined in samples drawn up to 11 days after exposure. The high S/N ratio (500) observed for the GB-2-DMAMP signal in the 11day sample poses the potential for an extended time frame of months for analysis with this new method for the retrospective detection of sarin exposure. To the best of our knowledge, this is the first report on LC–MS/MS trace analysis of regenerated GB from biological matrices.

The human respiratory system is a highly complex matrix that exhales many volatile organic compounds (VOCs). Breath-exhaled VOCs are often "unknowns" and possess low concentrations, which make their analysis, peak digging and data processing challenging. We report a new methodology, applied in a proof-of-concept experiment, for the detection of VOCs in breath. For this purpose, we developed and compared four complementary analysis methods based on solid-phase microextraction and thermal desorption (TD) tubes with two GC-mass spectrometer (MS) methods. Using eight model compounds, we obtained an LOD range of 0.02-20 ng/ml. We found that in breath analysis, sampling the exhausted air from Tedlar bags is better when TD tubes are used, not only because of the preconcentration but also due to the stability of analytes in the TD tubes. Data processing (peak picking) was based on two data retrieval approaches with an in-house script written for comparison and differentiation between two populations: sick and healthy. We found it best to use "raw" AMDIS deconvolution data (.ELU) rather than its NIST (.FIN) identification data for comparison between samples. A successful demonstration of this method was conducted in a pilot study (n = 21) that took place in a closed hospital ward (Covid-19 ward) with the discovery of four potential markers. These preliminary findings, at the molecular level, demonstrate the capabilities of our method and can be applied in larger and more comprehensive experiments in the omics world.

Long-term retrospective monitoring of exposure to organophosphorus nerve agents is challenging. We recently developed two highly sensitive analytical methods for regenerated sarin (GB) nerve agent in blood and its primary metabolite, isopropyl-methylphosphonic acid (IMPA), in urine. These methods were implemented in a toxicokinetics study carried out with sarin injected (i.v.) to rabbits at doses corresponding to 0.1, 0.5 or 0.9 LD50. The time frame for monitoring regenerated sarin from blood was 70 days for 0.1 LD50 and 0.5 LD50 and 77 days for 0.9 LD50, where rapid elimination occurred in the first 8 days with an initial average half-life of 1.2 days, followed by a second, slower elimination, with a terminal average half-life of 8.4 days. The time frame for monitoring IMPA in urine was 7, 15 and 16 days for 0.1 LD50, 0.5 LD50 and 0.9 LD50 intoxications, respectively. Rapid elimination of IMPA in urine occurred after exposure, with an average half-life of ~ 0.8 days on days 2–6. For the first time, a slower elimination route for IMPA, with an average half-life of ~ 4 days from day 6 onwards, was revealed. Both IMPA and regenerated sarin pharmacokinetics exhibit linearity with dose. The overlaid pharmacokinetic profiles of regenerated sarin in blood along with IMPA in urine emphasize the dominance of IMPA with a rapid decay in urine in the first week and the slower long-term decay of protein-bound sarin later in blood. To our knowledge, the two new sensitive methods exhibit the longest monitoring time frame reported in biological samples.

We present a simple method for chiral separation and analysis of organophosphorus nerve agents and apply it to monitor the enantioselective blood elimination kinetics of sarin in-vitro. The method is implemented in standard reverse phase LC-MS operating conditions, relieving the user of the dedicated operating conditions frequently demanded in chiral LC-MS analysis. The method consists of formation of diastereomers by a rapid derivatization with (R)-2-(1 aminoethyl) phenol, followed by LC-MS/MS analysis. Derivatization enantioselectivity was studied by comparing the reaction of optically pure sarin and racemic sarin, proving no substantial enantiomeric preference in the reaction and demonstrating the enantiomeric discrimination abilities of the technique. Enantioselective sarin elimination pathways were probed in-vitro by following the fast elimination kinetics of the two sarin enantiomers as well as its hydrolysis metabolite (isopropyl methyl-phosphonic acid, IMPA) in whole blood and plasma compared to water. Sarin enantiomers showed the known marked differences in elimination kinetics with rapid elimination of the (+) enantiomer and slower elimination of the (-) enantiomer in whole blood and plasma as well as dose-dependent kinetics (faster elimination at lower concentrations). We found that small amounts of acetonitrile in plasma prevent the rapid elimination of the (+) enantiomer, resulting in similar, slower elimination kinetics for both enantiomers.

Volatile metabolites in exhaled air have promising potential as diagnostic biomarkers. However, the combination of low mass, similar chemical composition, and low concentrations introduces the challenge of sorting the data to identify markers of value. In this paper, we report the development of pyAIR, a software tool for searching for volatile organic compounds (VOCs) markers in multi-group datasets, tailored for Thermal-Desorption Gas-Chromatography High Resolution Mass-Spectrometry (TD-GC-HRMS) output. pyAIR aligns the compounds between samples by spectral similarity coupled with retention times (RT), and statistically compares the groups for compounds that differ by intensity. This workflow was successfully tested and evaluated on gaseous samples spiked with 27 model VOCs at six concentrations, divided into three groups, down to 0.3 nL/L. All analytes were correctly detected and aligned. More than 80% were found to be significant markers with a p-value < 0.05; several were classified as possibly significant markers (p-value < 0.1), while a few were removed due to background level. In all group comparisons, low rates of false markers were found. These results showed the potential of pyAIR in the field of trace-level breathomics, with the capability to differentially examine several groups, such as stages of illness.

Phosphonic acids are the direct and immediate metabolites of organophosphorus chemical warfare agents (OP-CWAs). Accordingly, their detection serves for evaluating exposure to OP-CWAs in a terror or war scenario. After exposure, phosphonic acids are present in the blood; however, blood drawing must be carried out by medical personnel, hence the number of samples that can be drawn in a mass-casualty event is limited. Herein, we describe a new approach developed for the determination of phosphonic acids in blood using Dry Blood Spots (DBSs) on a filter paper. The method is based on a simple sample preparation protocol, followed by LC-MS-MS targeted (MRM) analysis. The detection limits of Soman (GD), Cyclosarin (GF) and VX metabolites in whole blood were as low as 1 ng/ml, while the detection limits were 0.3 ng/ml for the GF metabolite and 0.5 ng/ml for the Sarin (GB) metabolite. Good recoveries were obtained in the range of 1–100 ng/ml for GB and GD metabolites, and 3–100 ng/ml for GF, VX and RVX metabolites, with a linear response (R2 = 0.99). The method has proven to be reliable even with DBS samples stored up to 35 days at room temperature before analysis. This method was implemented in a 24 h time-course determination of the Sarin metabolite in an in - vivo experiment, after rat exposure to 1 LD50 of Sarin. This technique is simple, rapid, sensitive, robust, long lasting and compatible with field collection and storage; hence, it can serve for large-scale sampling and reliable monitoring of potential OP-CWAs casualties. Since DBS sampling is amenable to nonprofessionals, including self-sampling, this technique is highly suitable for mass-casualty incidents.

The ability of mono N-methyl-1,4-diazabicyclo[2.2.2]octane fluoride (Me-DABCOF, 1) to act as a bifunctional reagent that effectively and universally neutralizes both the persistent and extremely toxic blister agent HD and the nerve agent VX in nearly neutral aqueous solution, alumina powder or a hydrogel formulation, is described.

Nearly half a million people die annually due to mosquito-borne diseases. Despite aggressive mosquito population-control efforts, current strategies are limited in their ability to control these vectors. A better understanding of mosquito metabolism through modern approaches can contribute to the discovery of novel metabolic targets and/or regulators and lead to the development of better mosquito-control strategies. Currently, cutting-edge technologies such as gas or liquid chromatography-mass spectrometry-based metabolomics are considered 'mature technologies' in many life-science disciplines but are still an emerging area of research in medical entomology. This review primarily discusses recent developments and progress in the application of mass spectrometry-based metabolomics to answer multiple biological questions related to mosquito metabolism.

An analytical technique was developed for the detection of phosphorus-containing compounds, using high-resolution-accurate-mass (HRAM) LC-MS. This method was constructed as a “flagging” approach for chromatographic peaks of compounds suspected of containing phosphorus, using high-energy in-source CID fragmentation, in parallel with a Full-MS experiment. Selective peak picking is achieved by tracking the highly resolved low m/z product ions of phosphorus-containing products, indicative of several phosphate and phosphonate groups. The chemical formula is then specifically assigned by a simultaneous accurate mass measurement of the precursor ion, together with the confirmed presence of a phosphorus atom in the suspect peak. This new method is demonstrated with various phosphorus-containing compounds such as V-class chemical warfare agents, nerve agents’ degradation products, pesticides, organic phosphates and a phospholipid. For these compounds, the developed method was found to be effective at concentrations as low as 1 ng/mL, in environmental extracts of soil and in urine (in the case of the phospholipid), making it comparable to selective phosphorus detectors such as GC-FPD, but with a much broader scope for charged, polar, or larger compounds.

Although linked to a number of adverse health effects, studies of naphthalene are lacking in the exposure-relevant literature. This compound, a major by-product of the incomplete combustion of most organic materials (traffic vehicle exhaust, power generation, tobacco smoke, etc.), has characteristics that are in between those of volatile and semivolatile organic compounds (VOCs and SVOCs) and has consequently often been excluded from studies of both compound classes. A new, highly quantitative, rapid and spatially well-resolved micro-sampling method was developed for the sampling of naphthalene vapours in indoor, outdoor and personal air. The method is based on GC capillary columns as air samplers, solvent extraction and GC-MS analysis. For the first time, naphthalene concentrations were quantitatively determined at the surface level (around the boundary layer) and at 1 mm height intervals above an emitting surface. Minimal sampling flow rates of 5 mL min−1 were applied to reduce any geometrical or dynamical disturbance to the environmental air flow regime to a minimum. Toluene was highly efficient, precise and easy to apply in the extraction of the target compound from the GC column sampler. Method quantification was confirmed using a unique calibrated vapour generator based on an inert GC inlet. Using this source, a 13 ng L−1 limit of quantitation (LOQ) with 90% mass recovery over a linear dynamic range of five orders of magnitude in high precision and accuracy of ±13% and 35%, respectively, were determined and validated. The applicability and flexibility of the method was demonstrated by investigating the vapour concentration profiles generated “0”–20 mm above a naphthalene-emitting surface in outdoor air. The concept introduced in this research could be easily adopted for the reliable, sensitive and quantitative monitoring of other VOCs and SVOCs in different micro-spaces for various applications.

Negative electrospray ionization tandem mass spectrometry (ESI-MS/MS) is the first choice for detecting and identifying organophosphorus acids such as alkyl methylphosphonic acids, dialkyl phosphates and dialkyl thiophosphates. However, chemical noise in the lower mass regions, ion suppression and poor chromatographic retention of small, highly polar acids render the identification of such acids difficult, especially in complex matrices. To address this difficulty, we evaluated charge- reversal derivatizations of several polar organophosphorus acids with eight commercially- available, mostly pyridine-based reagents, possessing high proton-affinity amines. A simple and generic derivatization procedure of such acids in the presence of potassium carbonate and 18-crown-6 in acetonitrile was developed. After derivatization, the samples were analysed by LC-ESI(+)-MS/MS, and a fragmentation study was carried out. For all derivatizing agents, the resulting acid-derivatives were highly retained by LC and well responded in the ESI⿿MS/MS in the positive-ion mode. However, for several reagents, structural information losses were observed in MS/MS. Interestingly, each derivatizing agent exhibited a different MS/MS behaviour towards the analytes. Some derivatizing agents underwent considerable collision-induced dissociation exclusively at the amine tag portion, while others dissociated at the analyte portion and at the amine portion, depending on the alkyl side chain linked to the oxygen. Only the 3- (bromoacetyl)pyridine derivatives were highly specific and provided rich informative MS/MS spectra. The MS/MS identifications were based on characteristic neutral losses, diagnostic ions for the organophosphorus skeleton, and amine tag-characteristic product ions. This derivatization is beneficial in the identification of target and unknown organophosphorus acids.

The chromatograms obtained from the gas chromatography-electron ionization mass spectrometric (GC-EI-MS) analysis of extracts containing G-nerve agents in the presence of diesel, gasoline, etc., are dominated by hydrocarbon backgrounds that "mask" the G-nerve agents, leading to severe difficulties in identification. This paper presents a practical solution for this challenge by transferring the G-nerve agents from the organic phase into the aqueous phase using liquid-liquid extraction (LLE), followed by derivatization with 2-[(dimethylamino)methyl]phenol (2-DMAMP), allowing ultrasensitive LC-ESI-MS/MS analysis of the G-derivatives. The proposed approach enables rapid identification of trace amounts of G-nerve agents with limits of identification (LOIs) at the pg/mL scale.

A highly sensitive method for the detection and identification of sarin (GB), soman (GD) and cyclosarin (GF) chemical warfare agents (CWAs) in environmental outdoor and indoor matrices such as soil, asphalt, linoleum, formica, concrete and cloth was developed. The method incorporates derivatization of the G-type nerve agent extracts with 2-[(dimethylamino)methyl]phenol (2-DMAMP), followed by LC-ESI(+)-MS/MS analysis. Four LC-amenable extraction solvents were explored in terms of their extraction efficiency and the reaction rate of the derivatizing agent. The reaction time, temperature and derivatization reagent amount were optimized. The optimal procedure was found to be extraction with water by agitation (2 min), followed by the addition of 2-DMAMP directly into the injection vial and stirring for 5 min prior to LC-ESI(+)-MS/MS analysis, without any other pretreatment. The method was applied to real-world samples and exhibited very low detection limits (LODs) of 0.8–20 pg/cm2 in asphalt, linoleum, cloth, formica and concrete and 4 pg/g in soil. The newly developed method demonstrated significantly superior sensitivity compared to conventional GC–MS- and LC–MS-based methods for the identification of G-nerve agents and allowed the determination of both G-nerve agents and their hydrolysis products within a single LC–MS/MS run. The proposed methodology may be practical for verifying contaminated matrices collected in the battlefield or terror scenes in forensic investigations where trace level analysis is required.

Inorganic metal oxides have shown potential as matrices for assisting in laser desorption ionization with advantages over the aromatic acids typically used. Rhenium and tungsten oxides are attractive options due to their high work functions and relative chemical inertness. In this work, it is shown that ReO3 and WO3 , in microparticle (μP) powder forms, can efficiently facilitate ionization of various types of small molecules and provide minimized background contamination at analyte concentrations below 1 ng/µL. This study shows that untreated inorganic WO3 and ReO3 particles are valid matrix options for detection of protonatable, radical, and precharged species under laser desorption ionization. Qualitatively, the WO3 μP showed improved detection of apigenin, sodiated glucose, and precharged analyte choline, while the ReO3 μP allowed better detection of protonated cocaine, quinuclidine, ametryn, and radical ions of polyaromatic hydrocarbons at detection levels as low as 50 pg/µL. For thermometer ion survival yield experiments, it was also shown that the ReO3 powder was significantly softer than α-cyano-4-hydroxycinnaminic acid. Furthermore, it provided higher intensities of cocaine and polyaromatic hydrocarbons, at laser flux values equal to those used with α-cyano-4-hydroxycinnaminic acid.

A novel analytical technique for the structural elucidation of compounds bearing a tertiary amine side chain via "in vial" instantaneous oxidation and liquid chromatography mass spectrometry (LC-MS) was developed. A series of lidocaine homologs and benzimidazole derivatives with a major/single amine representative base peak in both their EI-MS and ESI-MS/MS spectra were subjected to oxidation by a 0.1% solution of hydrogen peroxide (including several 16 O/18 O exchange experiments), followed by LC-ESI-MS/MS analysis. The N-oxide counterparts promoted extensive fragmentation with complete coverage of all parts of the molecule, enabling detailed structural elucidation and unambiguous identification of the unoxidized analytes at low nanogram per milliliter levels.

A methodology for sensitive determination of sarin (GB), soman (GD) and cyclosarin (GF) chemical warfare agents in aqueous media was developed. The method incorporates direct derivatization with 2-[(dimethylamino)methyl]phenol (2-DMAMP), a commercially available, water-soluble reagent, followed by LC-ESI-MS/MS analysis in the positive ion mode. Five derivatization agents were characterized for their MS/MS fragmentation pattern, and their reaction time, temperature and derivatization-reagent amount were optimized. The developed derivatization reaction is simple, fast (1min) and proceeds at ambient temperature. Sample preparation consists of only the addition of the reagent directly into an injection vial prior to LC-ESI(+)-MS/MS analysis. All 2-DMAMP derivatives were stable for at least 48h and had unique tandem mass spectra characterized by common product ions at m/z 230 and 185. Compared with conventional GC-MS or LC-MS methods, simplicity, better sensitivity and informative MS/MS spectra were achieved by this method. Limits of detection (LODs), identification (LOIs), and quantification (LOQs) were determined in tap water and found to be 1pg/ml, 4pg/ml and 4pg/ml respectively. The proposed methodology is appropriate for routine evaluation of contaminated water supplies and has the advantage of a simultaneous analysis of both derivatized G-nerve agents and their intact hydrolysis products within a single LC-MS analysis.

Dry blood spot (DBS), a micro whole-blood sampling technique, enables rapid and self-blood collection; it is stable and economical. Currently, DBS filters require various sample preparation procedures specifically tailored for the target compounds, which are followed by GC–MS or LC–MS analysis. However, the small amounts of blood make the approach analytically challenging, mostly in terms of sensitivity and quantification. Herein, we introduce a new DBS concept for GC-compatible volatile to semi-volatile compounds in which DBS is directly coupled with thermal desorption analysis, thus eliminating time consuming treatments. Furthermore, to stabilize the target compound over the sampling DBS substrate, a commercial filter based on an extremely efficient trapping adsorption phase, styrene–divinylbenzene (SDVB), is first used. The performance of the new SDVB–DBS concept was demonstrated herein for monitoring the most volatile chemical warfare agent, sarin, which might be present in blood and the detection of which is usually challenging due to its rapid metabolism. This study encompasses adequate sampling and analysis method parametrization and validation, leading to a detection sensitivity of 100 pg sarin per 30 µL whole blood in 5-day-old samples, with a linear dynamic range of two orders of magnitude, adequate precision, and acceptable accuracy. Applying the method to an in-vivo mouse intranasal exposure experiment (3LD50 GB) enabled the successful detection of 25–90 ng mL−1 free sarin in blood samples drawn 2 min after exposure. The method’s performance clearly emphasizes the potential of the new concept in “freezing the clock” for reactive whole blood media in pharmacokinetics and pharmacodynamics studies, as well as in applications in which informative and reliable monitoring of unstable target compounds and biomarkers is desired.

The use of chemical warfare agents has become an issue of emerging concern. One of the challenges in analytical monitoring of the extremely toxic ‘V’-type chemical weapons [O-alkyl S-(2-dialkylamino)ethyl alkylphosphonothiolates] is to distinguish and identify compounds of similar structure. MS analysis of these compounds reveals mostly fragment/product ions representing the amine-containing residue. Hence, isomers or derivatives with the same amine residue exhibit similar mass spectral patterns in both classical EI/MS and electrospray ionization-MS, leading to unavoidable ambiguity in the identification of the phosphonate moiety. A set of five ‘V’-type agents, including O-ethyl S-(2-diisopropylamino)ethyl methylphosphonothiolate (VX), O-isobutyl S-(2-diethylamino)ethyl methylphosphonothiolate (RVX) and O-ethyl S-(2-diethylamino)ethyl methylphosphonothiolate (VM) were studied by liquid chromatography/electrospray ionization/MS, utilizing a QTRAP mass detector. MS/MS enhanced product ion scans and multistage MS3 experiments were carried out. Based on the results, possible fragmentation pathways were proposed, and a method for the differentiation and identification of structural isomers and derivatives of ‘V’-type chemical warfare agents was obtained. MS/MS enhanced product ion scans at various collision energies provided information-rich spectra, although many of the product ions obtained were at low abundance. Employing MS3 experiments enhanced the selectivity for those low abundance product ions and provided spectra indicative of the different phosphonate groups. Study of the fragmentation pathways, revealing some less expected structures, was carried out and allowed the formulation of mechanistic rules and the determination of sets of ions typical of specific groups, for example, methylphosphonothiolates versus ethylphosphonothiolates. The new group-specific ions elucidated in this work are also useful for screening unknown ‘V’-type agents and related compounds, utilizing precursor ion scan experiments. Copyright © 2013 John Wiley & Sons, Ltd.

Understanding protein tertiary and quaternary structures is crucial to a better understanding of their biological functions. Here we illustrate for tryptophan synthase that tandem mass spectrometry (MS/MS) reveals not only protein subunit architectures, but also protein unfolding behavior when coupled with ion mobility (IM). In the present study, we verified the subunit arrangement with surface induced dissociation (SID). We are able to correlate experimental results by IM with those obtained in unfolding simulations for the hetero-tetramer Tryptophan Synthase (TS) protein complex by identifying the presence of at least three stable intermediates (I1, I2, and I3) during the unfolding process in collision induced dissociation (CID). We illustrate that the unfolding of the TS complex is likely due to the initial unfolding of an α-monomer subunit, followed by the unfolding of the second α-monomers. We also illustrate the ability of this combination of techniques to not only identify conformational changes of TS upon addition of D,L-α-glycerol phosphate (GP), but also to determine the location of the ligand, which is buried within the α-monomer of the TS.

Glu, Gln, Pro, and Ala are the main amino acids involved in ammonia detoxification in mosquitoes. In order to develop a tandem mass spectrometry method (MS(2)) to monitor each carbon of the above isotopically-labeled (13)C-amino acids for metabolic studies, the compositions and origins of atoms in fragments of the protonated amino acid should be first elucidated. Thus, various electrospray (ESI)-based MS(2) tools were employed to study the fragmentation of these unlabeled and isotopically-labeled amino acids and better understand their dissociation pathways. A broad range of fragments, including previously-undescribed low m/z fragments was revealed. The formulae of the fragments (from m/z 130 down to m/z 27) were confirmed by their accurate masses. The structures and conformations of the larger fragments of Glu were also explored by ion mobility mass spectrometry (IM-MS) and gas-phase hydrogen/deuterium exchange (HDX) experiments. It was found that some low m/z fragments (m/z 27-30) are common to Glu, Gln, Pro, and Ala. The origins of carbons in these small fragments are discussed and additional collision induced dissociation (CID) MS(2) fragmentation pathways are proposed for them. It was also found that small fragments (≤m/z 84) of protonated, methylated Glu, and methylated Gln are the same as those of the underivatized Glu and Gln. Taken together, the new approach of utilizing low m/z fragments can be applied to distinguish, identify, and quantify (13)C-amino acids labeled at various positions, either in the backbone or side chain.

The quaternary structure of proteins determines their biological function, and a majority of proteins exist as oligomers in vivo with miscellaneous architectures. Mass spectrometry (MS) can be applied to study the stoichiometry and interactions of protein complexes by molecular weight measurements under gentle instrumental conditions where noncovalent interactions are preserved in the gas phase. Recently, there have been extensive efforts to also utilize ion mobility (IM) techniques combined with MS for structural studies of noncovalent biological complexes, including virus assembly pathways, because IM provides conformational information, which is not accessible by MS, for these gas-phase ions. Experimentally measured collisional cross sections (CCSs) from IM can serve as constraints for architecture determination by molecular modeling. Additionally, tandem MS can be used to dissociate gas-phase complexes. A protein assembly would ideally dissociate into various noncovalent subcomplexes, and the topology of the original complex could be derived by piecing together all the subcomplex products. The common tandem MS method, collision induced dissociation (CID), involves activation of the complexes by collision with neutral gas atoms or molecules. Typically, CID results in an “asymmetric” dissociation into highly charged monomers and complementary (n 1)-mers (although a few exceptions have been reported), and studies have suggested that unfolding of protein complexes occurs in CID. It is therefore difficult to relate the CCS measurements of CID product ions to the complexes native structure. Tandem MS can alternatively be achieved by surface induced dissociation (SID) where the complexes collide with a surface target. Previous research in our group has shown that several protein complexes dissociate in a more “symmetric” manner with SID than by CID, and have charge distributed more proportionally to the mass. We hypothesized that dissociation might occur in the absence of gradual monomer unfolding for SID because activation by SID is a single-step, higher-energy deposition, fast process that is different from the multistep, slower CID process. SID has recently been applied to determining the quaternary structure of a heterohexameric protein with information from subunit product ions such as heterotrimers unique to SID. We present herein the first IM measurements on the SID products of several protein complexes, along with comparison to CID products, by using a modified quadrupole/IM/time-offlight (Q/IM/TOF) instrument. Briefly, the precursor ions are dissociated by CID or SID cells placed in front of the IM cell. The product ions are subsequently separated based on their size, shape, and charge under the influence of a continuous series of electrical pulses and friction with neutral gas in the IM cell. The drift times of the ions are recorded, with larger and lower-charged ions experiencing longer drift times. Experimental CCSs can be derived from the measured drift times and mass-to-charge-ratios. Theoretical CCSs can be calculated from crystal structures. Nativelike ions should have experimental CCSs similar to the crystal structure, whereas unfolded ions are expected to show larger CCS values because of an increased surface area. We first examined the remaining undissociated pentamer precursor of C-reactive protein (CRP) after activation by either CID or SID. Triethylammonium acetate (TEAA) was added in the electrospraying buffer, which has been reported as a charge reducing additive to increase the stability of protein complexes in the gas phase. Without TEAA no remaining precursor could be observed in SID, even at low acceleration voltages. The addition of TEAA did not cause any remarkable structural change of the protein as the CCSs of the precursor did not change after charge reduction. The + 18 precursor of CRP was selected and activated. Examination of precursor CCSs at various SID acceleration voltages (20–50 V) reveals that most of the CRP pentamer dissociated without extensive increase in CCS of the remaining precursor. In CID, however, the CCS of the undissociated CRP pentamer first decreased at low acceleration voltages and then increased considerably above its dissociation threshold of about 80 V. It reached a stable unfolding intermediate with CID around 100 V, where the CCS does not additionally increase with increasing CID acceleration voltages (data not shown). It is impractical to determine one acceleration voltage at which the amounts of the internal energies deposited in CID and SID are identical because of different mechanisms and complications from the physical properties of large protein complexes. Nonetheless, we show here a representative comparison between CID at 100 V (Figure 1, top right) and SID at 40 V (Figure 1, bottom [*] M. Zhou, Dr. S. Dagan, Prof. V. H. Wysocki Department of Chemistry and Biochemistry, University of Arizona 1306 E. University Blvd., Tucson, AZ (USA) E-mail: vwysocki@email.arizona.edu [] Permanent address: Israel Institute for Biological Research (IIBR) POB 19, Ness Ziona 74100 (Israel)

We report a systematic empirical investigation of ESI(+)-MS-MS dissociation pathways of over 1000 spectra of small organic compounds, containing more than 30 chemical functional groups. The dissociation processes of the protonated molecular ions were explored and interpretated. We derived typical basic fragmentation channels for individual functional groups and established a unified set of fragmentation rules. Multiple bond cleavages of molecules containing single and multiple functional groups were explored as well and the corresponding fragmentation rules were derived. Applying these rules enabled to match between proposed chemical structures and an ESI(+)-MS-MS spectrum of an “unknown”. Comparison to EI fragmentation routes was also carried out. Despite the general dissimilarity between ESI(+)-MS-MS and EI-MS spectra, we exploit the minor similarities between the spectra, and utilizing NIST-EI database and search option, can be successfully reduced the number of proposed structures. The two step methodology developed here is demonstrated and evaluated in the identification of various “unknowns”.

The degradation of the warfare agent sulfur mustard (HD) adsorbed onto KF/Al(2)O(3) sorbents is described. These processes were explored by MAS NMR, using (13)C-labeled sulfur mustard (HD*) and LC-MS techniques. Our study on the detoxification of this blister agent showed the formation of nontoxic substitution and less-toxic elimination products (t(1/2) = 3.5-355 h). Interestingly, the reaction rates were found to be affected by MAS conditions, i.e., by a centrifugation effect. The products and the mechanisms of these processes are discussed.

The use of silicon nanoparticles for laser desorption/ionization (LDI) is a new appealing matrix-less approach for the selective and sensitive mass spectrometry of small molecules in MALDI instruments. Chemically modified silicon nanoparticles (30 nm) were previously found to require very low laser fluence in order to induce efficient LDI, which raised the question of internal energy deposition processes in that system. Here we report a comparative study of internal energy deposition from silicon nanoparticles to previously explored benzylpyridinium (BP) model compounds during LDI experiments. The internal energy deposition in silicon nanoparticle-assisted laser desorption/ionization (SPALDI) with different fluorinated linear chain modifiers (decyl, hexyl and propyl) was compared to LDI from untreated silicon nanoparticles and from the organic matrix, α-cyano-4-hydroxycinnamic acid (CHCA). The energy deposition to internal vibrational modes was evaluated by molecular ion survival curves and indicated that the ions produced by SPALDI have an internal energy threshold of 2.8–3.7 eV. This is slightly lower than the internal energy induced using the organic CHCA matrix, with similar molecular survival curves as previously reported for LDI off silicon nanowires. However, the internal energy associated with desorption/ionization from the silicon nanoparticles is significantly lower than that reported for desorption/ionization on silicon (DIOS). The measured survival yields in SPALDI gradually decrease with increasing laser fluence, contrary to reported results for silicon nanowires. The effect of modification of the silicon particle surface with semifluorinated linear chain silanes, including fluorinated decyl (C10), fluorinated hexyl (C6) and fluorinated propyl (C3) was explored too. The internal energy deposited increased with a decrease in the length of the modifier alkyl chain. Unmodified silicon particles exhibited the highest analyte internal energy deposition. These findings may suggest a role of the modifier as a moderator in the energy dissipation and relaxation process. The relatively low internal energy content of SPALDI-produced ions indicates that this is a “soft” desorption technique, with potential advantages in the analysis of labile compounds.

A novel in-line surface-induced dissociation (SID) device was designed and implemented in a commercial QTOF instrument (Waters/Micromass QTOF II). This new setup allows efficient SID for a broad range of molecules. It also allows direct comparison with conventional collision-induced dissociation (CID) on the same instrument, taking advantage of the characteristics of QTOF instrumentation, including extended mass range, improved sensitivity, and better resolution compared with quadrupole analyzers and ion traps. Various peptides and a noncovalent protein complex have been electrosprayed and analyzed with the new SID setup. Here we present SID of leucine enkephalin, fibrinopeptide A, melittin, insulin chain-B, and a noncovalent protein complex from wheat, heat shock protein 16.9. The SID spectra were also compared to CID spectra. With the SID setup installed, ion transmission proved to be efficient. SID fragmentation patterns of peptides are, in general, similar to CID, with differences in the relative intensities of some peaks such as immonium ions, backbone cleavage b- versus y-type ions, and y- versus y-NH3 ions, suggesting enhanced accessibility to high-energy/secondary fragmentation channels with SID. Furthermore, these results demonstrate that the in-line SID setup is a valid substitute for CID, with potential advantages for activation of singly/multiply charged peptides and larger species such as noncovalent protein complexes.

We present a unique class of polythiacrown macro- and gigantocyclic[9] systems, consisting of ethylene 1,2-dithioglycol (ETG) to poly(ethylene thioglycol) (ETGn) bridges over one to six diacetal units of the cis-1,3,5,7-tetraoxadecalin (TOD) type. The latter is a dissymmetric, chiral moiety, incorporating a cavity with built-in high electron lone pair concentration, serving as the “core” of chiral macrocyclic host systems with good inclusion ability of ions and polar molecules. We describe two approaches: (i) the reactions of the 2,6-bis(bromomethyl)-cis-TOD podand (6) with ETG or higher ETGns (12n), in Cs2CO3 promoted processes, leading to the innate but uncontrolled formation of polythiacrown-TOD macrocycles having ETG/TOD ratios of 1:1 (7), 2:2 (8) and further 3:3–6:6 (111/m)10 macrocycles via open dithiol intermediates, and (ii) judicious preparation, using K2CO3, of oligomeric dibromide intermediates with ETGn:TOD ratios 1:2, 2:3 or 3:4 (14n/m), which led (with further ETGn) in a controlled way to the 2:2 (8n), or 3:3, 4:4 and 6:6 (11n/m) macro- and gigantocyclic systems. Altogether, the outcome of these processes depends on the relative concentrations of the reactants. Synthesis was accompanied by detailed (NMR and MS) spectroscopy. X-ray crystallographic analysis of a number of macrocycles, complemented by (MM & MD) computation, made possible valuable structural, stereochemical and conformational analysis. While sophisticated in their stereochemical features, these systems are readily prepared in enantiopure form and hold great promise of chemical reactivity in metal ion inclusion and molecular and chiral recognition.(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2007)

VX is one of the most toxic chemical warfare agents. Its low volatility and its persistence in the environment raise the issue of long-term exposure risks, either by inhalation or by transdermal penetration. Therefore, a topic of acute interest is the fate of VX in preservative environmental surfaces. In this work, the fate of VX in asphalt pavement, a suspected preservative matrix, was explored, by applying a novel quantitative method for the extraction of trapped VX from "digested" asphalt. It is based on dissolution of asphalt in toluene, precipitation of the heavy components by basic methanol followed by GC-NPD analysis. This method is complementary to methanol extraction of VX from the outer surface of asphalt, and enabled us to explore the total amount of viable VX both on and inside the matrix. Using this method, bis-diisopropylaminoethyl-disulfide [(DES)2], a degradation product of VX, was also assayed. Small chunks of Asphalt were spiked with VX, sealed and analyzed after various aging periods up to 425 days. The level of VX on the outer surface of the asphalt was found to be diminishing with time following a single-exponential decay. The level inside the asphalt increases during the first day, decays steeply to a level of about 5% during the following two weeks, and declines moderately during all the period up to 425 days following a bi-exponential decay. The total recovery of VX from the asphalt declined from almost 100% after 30 minutes to about 2% after 425 days, with a half-life of about 14 days.

The complexity of a matrix is in many cases the major limiting factor in the detection and identification of trace level analytes. In this work, the ability to detect and identify trace level of pesticides in complex matrices was studied and compared in three, relatively new methods: (a) GC–PFPD–MS where simultaneous PFPD (pulsed flame photometric detection) and MS analysis is performed. The PFPD indicates the exact chromatographic time of suspected peaks for their MS identification and provides elemental information; (b) automatic GC–MS data analysis using the AMDIS (“Automated Mass Spectral Deconvolution and Identification System”) software by the National Institute of Standards and Technology; (c) GC–MS–MS analysis. A pesticide mixture (MX-5), containing diazinon, methyl parathion, ethyl parathion, methyl trithion and ethion was spiked, in descending levels from 1 ppm to 10 ppb, into soil and sage (spice) extracts and the detection level and identification quality were evaluated in each experiment. PFPD–MS and AMDIS exhibited similar performance, both superior to standard GC–MS, revealing and identifying compounds that did not exhibit an observable GC peak (either buried under the chromatographic background baseline or co-eluting with other interfering GC peaks). GC–MS–MS featured improved detection limits (lower by a factor of 6–8) compared to AMDIS and PFPD–MS. The GC–PFPD–MS–MS combination was found useful in several cases, where no reconstructed ion chromatogram MS–MS peaks existed, but an MS–MS spectrum could still be extracted at the elution time indicated by PFPD. The level of identification and confirmation with MS–MS was inferior to that of the other two techniques. In comparison with the soil matrix, detection limits obtained with the loaded sage matrix were poorer by similar factors for all the techniques studied (factors of 5.8, >6.5 and 4.0 for AMDIS, PFPD–MS and MS–MS, respectively). Based on the above results, the paper discusses the trade-offs between detectivity and identification level with the compared three techniques as well as other more traditional techniques and approaches.

A new method for the fast screening of cocaine and 6-monoacetylmorphine (6-MAM) in a single hair, using gas chromatography/mass spectrometry (GC/MS), is described. The analyses are conducted in less than 10 min with minimal sample preparation. The novel method combines the ChromatoProbe direct sample introduction device for intrainjector thermal extraction, fast GC separation, a supersonic molecular beam GC/MS interface and hyperthermal surface ionization (HSI). The technique has been successfully employed for the detection of cocaine in as little as a 1-mm section of hair using selected ion monitoring (SIM). Unambiguous full scan mass spectra of cocaine and 6-MAM were obtained on a single hair for cocaine and heroin users, respectively. HSI was found to be almost 3 orders of magnitude more selective than electron impact ionization for cocaine compared with the major hair constituents, with a minimum detected concentration of approximately 10 ppb in the SIM mode. Results obtained for 12 drug users showed full qualitative agreement with similar results using rigorous solvent extraction followed by electrospray-liquid chromatography/mass spectrometry analysis. However, quantitative studies showed only partial agreement. No false positives were observed for 10 drug free subjects. This method enables fast drug monitoring along the hair length which permits time correlation studies.

A novel method for fast analysis is presented. It is based on laser desorption injection followed by fast gas chromatography-mass spectrometry (GC-MS) in supersonic molecular beams. The sample was placed in an open air or purged laser desorption compartment, held at atmospheric pressure and near room temperature conditions. Desorption was performed with a XeCl Excimer pulsed laser with pulse energy of typically 3 mJ on the surface. About 20 pulses at 50 Hz were applied for sample injection, resulting in about 0.4 s injection time and one or a few micrograms sample vapor or small particles. The laser desorbed sample was further thermally vaporized at a heated frit glass filter located at the fast GC inlet. Ultrafast GC separation and quantification was achieved with a 50-cm-long megabore column operated with a high carrier gas flow rate of up to 240 mL/min. The high carrier gas flow rate provided effective and efficient entrainment of the laser desorbed species in the sweeping gas. Following the fast GC separation, the sample was analyzed by mass spectrometry in supersonic molecular beams. Both electron ionization and hyperthermal surface ionization were employed for enhanced selectivity and sensitivity. Typical laser desorption analysis time was under 10 s. The laser desorption fast GC-MS was studied and demonstrated with the following sample/matrices combinations, all without sample preparation or extraction: (a) traces of dioctylphthalate plasticizer oil on stainless steel surface and the efficiency of its cleaning; (b) the detection of methylparathion and aldicarb pesticides on orange leaves; (c) water surface analysis for the presence of methylparathion pesticide; (d) caffeine analysis in regular and decaffeinated coffee powder; (e) paracetamol and codeine drug analysis in pain relieving drug tablets; (f) caffeine trace analysis in raw urine; (g) blood analysis for the presence of 1 ppm lidocaine drug. The features and advantages of the laser desorption fast GC-MS are demonstrated and discussed.

The features of supersonic molecular beams (SMB) are used to improve gas chromatography—mass spectrometry (GC–MS) performance and establish a new method of fast GC–MS. In SMB, the sample compounds are vibrationally cooled such that their electron impact mass spectra are characterized by enhanced M+ peaks, together with library-searchable fragments. A new ionization method, hyperthermal surface ionization (HSI), provides ultrasensitive ionization coupled with a tunable degree of selectivity for nitrogen-containing drugs. SMB enables the use of very high carrier gas flow rates which, when a short megabore column is used, results in ultra-fast GC–MS having conventional chromatographic peak widths. Thus, fast GC–MS in SMB can be performed with conventional quadrupole analyzers. The slightly reduced GC resolution can, in many cases, be compensated for by the selectivity of hyperthermal surface ionization or by the increased EI selectivity through enhanced M+. “Fast”, “very-fast”, and “ultra-fast” GC–MS are defined and demonstrated with drugs, and the ability to analyze underivatized steroids is shown. Practical examples are shown including ultra-fast GC–MS of lidocaine in human plasma extract achieved in a few seconds and screening of other drugs, without any sample preparation or extraction, achieved in less than three minutes. We conclude that GC–SMB–MS exhibits a combination of faster analysis with improved sensitivity and selectivity, a wider range of molecules amenable to GC–MS, improved MS information, and higher a degree of flexibility.

The use of chemical warfare agents has become an issue of emerging concern. One of the challenges in analytical monitoring of the extremely toxic 'V'-type chemical weapons [O-alkyl S-(2-dialkylamino)ethyl alkylphosphonothiolates] is to distinguish and identify compounds of similar structure. MS analysis of these compounds reveals mostly fragment/product ions representing the amine-containing residue. Hence, isomers or derivatives with the same amine residue exhibit similar mass spectral patterns in both classical EI/MS and electrospray ionization-MS, leading to unavoidable ambiguity in the identification of the phosphonate moiety. A set of five 'V'-type agents, including O-ethyl S-(2-diisopropylamino)ethyl methylphosphonothiolate (VX), O-isobutyl S-(2-diethylamino)ethyl methylphosphonothiolate (RVX) and O-ethyl S-(2-diethylamino)ethyl methylphosphonothiolate (VM) were studied by liquid chromatography/electrospray ionization/MS, utilizing a QTRAP mass detector. MS/MS enhanced product ion scans and multistage MS(3) experiments were carried out. Based on the results, possible fragmentation pathways were proposed, and a method for the differentiation and identification of structural isomers and derivatives of 'V'-type chemical warfare agents was obtained. MS/MS enhanced product ion scans at various collision energies provided information-rich spectra, although many of the product ions obtained were at low abundance. Employing MS(3) experiments enhanced the selectivity for those low abundance product ions and provided spectra indicative of the different phosphonate groups. Study of the fragmentation pathways, revealing some less expected structures, was carried out and allowed the formulation of mechanistic rules and the determination of sets of ions typical of specific groups, for example, methylphosphonothiolates versus ethylphosphonothiolates. The new group-specific ions elucidated in this work are also useful for screening unknown 'V'-type agents and related compounds, utilizing precursor ion scan experiments.

Gas chromatography-mass spectrometry (GC-MS) analyses of thermally labile compounds have been studied by using a short column fast gas chromatograph, coupled with fly-through electron ionization in supersonic molecular beams. Thirty-two compounds, which include steroids, carbamate pesticides, antibiotic drugs, and other pharmaceutical compounds, have been analyzed and the details of their GC-MS analysis are provided. The ability to analyze thermally labile compounds is discussed in relation to the speed of analysis. A new term, “speed enhancement factor” (SEF), is defined as the product of column length reduction and the carrier gas linear velocity increase, as compared with normal GC-MS conditions. Fast, very fast, and ultra-fast GC-MS are defined with a SEF in the ranges of 5–30, 30–400, and 400–4000, respectively. Trade-offs in the degree of dissociation, speed, gas chromatograph resolution, and sensitivity were studied and examined with thermally labile molecules. The experimental factors that affect the dissociation are described with emphasis on its reduction. We claim that the use of supersonic molecular beams for sampling and ionization provides the ultimate capability in the GC-MS of thermally labile compounds. The obtained 70-eV electron ionization mass spectra are shown, and an enhanced relative abundance of the molecular ion is demonstrated together with library search capability of these mass spectra, which is better than that reported with particle beam liquid chromatography-mass spectrometry. The performance of fast GC-MS in supersonic molecular beams is compared with other methods of fast GC-MS and with particle beam liquid chromatography-mass spectrometry.

Thermal and hyperthermal surface ionization (SI) mass spectra of nicotine, caffeine and lidocaine were obtained using a rhenium oxide surface. Thermal surface ionization was studied on an oxidized surface positioned inside an electron impact ion source, while hyperthermal surface ionization (HSI) was obtained upon seeding the compounds into a hydrogen or helium supersonic molecular beam that scattered from the rhenium oxide surface. Both HSI and SI provide rich, informative and complementary mass spectral information. The results indicate that SI follows thermal dissociation processes on the surface prior to the desorption of the ion, while in HSI no thermal equilibrium is established and the ionization process is impulsive, followed by mostly unimolecular ion dissociation. HSI mass spectra are similar to electron impact mass spectra in the fragment ion masses, but the observed relative intensities are different. HSI is a softer ionization method compared to SI, and enables the degree of ion fragmentation to be tuned so that it can be minimized to a low level at low molecular kinetic energy. In SI, limited control over the degree of fragmentation is possible through the surface temperature. The analytical mass spectrometric applications of SI and HSI are briefly mentioned.

This work describes and evaluates the coupling of a fast gas chromatograph (GC) based on a short column and high carrier gas flow rate to a supersonic molecular beam mass spectrometer (MS). A 50 cm long megabore column serves for fast GC separation and connects the injector to the supersonic nozzle source. Sampling is achieved with a conventional syringe based splitless sample injection. The injector contains no septum and is open to the atmosphere. The linear velocity of the carrier gas is controlled by a by-pass (make-up) gas flow introduced after the column and prior to the supersonic nozzle. The supersonic expansion serves as a jet separator and the skimmed supersonic molecular beam (SMB) is highly enriched with the heavier organic molecules. The supersonic molecular beam constituents are ionized either by electron impact (EI) or hyperthermal surface ionization (HSI) and mass analyzed. A 1 s fast GC—MS of four aromatic molecules in methanol is demonstrated and some fundamental aspects of fast GC—MS with time limit constraints are outlined. The flow control (programming) of the speed of analysis is shown and the analysis of thermolabile and relatively non-volatile molecules is demonstrated and discussed. The tail-free, fast GC—MS of several mixtures is shown and peak tailing of caffeine is compared with that of conventional GC—MS. The improvement of the peak shapes with the SMB—MS is analyzed with the respect to the elimination of thermal vacuum chamber background. The extrapolated minimum detected amount was about 400 ag of anthracence- d 10 , with an elution time which was shorter than 2s. Repetitive injections could be performed within less than 10 s. The fast GC—MS in SMB seems to be ideal for fast target compound analysis even in real world, complex mixtures. The few seconds GC—MS separation and quantification of lead (as tetraethyllead) in gasoline, caffeine in coffee, and codeine in a drug is demonstrated. Controlled HSI selectivity is demonstrated in the range of 10 1 to 10 4 anthracene/decane which helped to simplify the selective analysis of aromatic molecules in gasoline. The contribution of SMB to the operation of the fast GC—MS is summarized and the compatibility with conventional GC having a megabore column is shown. Splitless injections of 100 μL sample solutions for trace level concentration detection is also presented (with a conventional GC).

Chemically modified silicon nanoparticles were applied for the laser desorption/negative ionization of small acids. A series of substituted sulfonic acids and fatty acids was studied. Compared to desorption ionization on porous silicon (DIOS) and other matrix-less laser desorption/ionization techniques, silicon nanoparticle-assisted laser desorption/ionization (SPALDI) mass spectrometry allows for the analysis of acids in the negative ion mode without the observation of multimers or cation adducts. Using SPALDI, detection limits of many acids reached levels down to 50 pmol/µl. SPALDI of fatty acids with unmodified silicon nanoparticles was compared to SPALDI using the fluoroalkyl silylated silicon powder, with the unmodified particles showing better sensitivity for fatty acids, but with more low-mass background due to impurities and surfactants in the untreated silicon powder. The fatty acids exhibited a size-dependent response in both SPALDI and unmodified SPALDI, showing a signal intensity increase with the chain length of the fatty acids (C12-C18), leveling off at chain lengths of C18-C22. The size effect may be due to the crystallization of long chain fatty acids on the silicon. This hypothesis was further explored and supported by SPALDI of several, similar sized, unsaturated fatty acids with various crystallinities. Fatty acids in milk lipids and tick nymph samples were directly detected and their concentration ratios were determined by SPALDI mass spectrometry without complicated and time-consuming purification and esterification required in the traditional analysis of fatty acids by gas chromatography (GC). These results suggest that SPALDI mass spectrometry has the potential application in fast screening for small acids in crude samples with minimal sample preparation. Copyright c � 2010 John Wiley & Sons, Ltd.

Cholesterol in a hydrogen-seeded supersonic molecular beam was scattered from a continuously oxidized rhenium foil. The hyperthermal surface scattering exhibited efficient molecular ionization with a controlled amount of molecular ion dissociation. At 5.3 eV incident molecular kinetic energy the hyperthermal surface ionization mass spectrum was dominated by the parent molecular ion. Upon the increase of the molecular kinetic energy, a gradual increase in the degree of ion dissociation was observed. At 22eV incident kinetic energy the parent ion was completely dissociated and the mass spectrum was dominated by an extensive consecutive fragmentation. An efficient kinetic-vibrational energy transfer was observed, and it is extimated to be over 18% of the available incident kinetic energy. The implication for surface collisionally-activated dissociation of polyatomic ions is discussed. Rhenium oxide is suggested as an optimal surface for this purpose, as well as for the hyperthermal surface ionization of neutral species.

Silicon nanopowder (5-50 nm) was applied as a matrix for the analysis of small molecules in laser desorption/ionization mass spectrometry. In contrast with conventional matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry, the matrix background interference in the low mass range was significantly reduced. Effects of the particle size and sample preparation procedures on the background mass spectra and the analyte signal intensity have been investigated, and an optimized powder and sample preparation protocol was established. Several surface characterization tools have been applied as well. Both positive mode and negative mode laser desorption/ionization have been applied to different analytes including drugs, peptides, pesticides, acids, and others. Detection limits down to the low femtomole per microliter levels were achieved for propafenone and verapamil drugs. The method developed was found relatively tolerant to salt contamination, which allowed the direct analysis of morphine and propaphenone in untreated urine and triazine herbicides in a soil extract. The new silicon-nanoparticle-assisted laser desorption ionization method was found to be highly selective, which may be due to analyte-dependent precharging in solution, prior to vacuum laser desorption. Some aspects of the charge-transfer mechanism have been studied and discussed. In comparison with standard MALDI matrixes, the silicon nanopowder requires much lower laser fluence (contributing to a reduced background) has much better surface homogeneity, and is more tolerant to salt interference, which makes it an easily applicable practical tool at a potentially low cost.

Previous gas-phase dissociation experiments of protein−protein complexes have resulted in product ion distributions that are asymmetric by charge and mass, providing limited insight into the chemical nature of subunit organization and interaction. In these experiments, a symmetric charge distribution results from an “energy sudden” collision of protein−protein complexes with a surface, indicating that it may be possible to probe the suboligomeric structure of noncovalent complexes in the gas phase. It is proposed that energy sudden surface activation of cytochrome C homodimers results in dissociation without significant unfolding of one of the monomeric subunits. Previously proposed mechanisms for the dissociation of protein−protein complexes are discussed in the context of these results. These experiments demonstrate the potential to preserve the structural details of subunit interaction within a protein−protein complex and help elucidate the asymmetric nature of macromolecular dissociation in the gas phase.

Charge states of noncovalent protein complexes in the gas phase are known to affect their propensity for unfolding and dissociation. In this work, C-reactive protein (CRP) pentamer and Concanavalin A (ConA) tetramer at different charge states were subjected to collision induced dissociation (CID) and surface induced dissociation (SID) in a modified quadrupole/ion mobility/time-of-flight mass spectrometer. Charge manipulation was achieved through solution addition of charge reducing (triethylammonium acetate) or supercharging (3-nitrobenzylalcohol) reagents. The results show that charge reduction increases the stability of the proteins to dissociation and suppresses unfolding of the precursors. While CID becomes less effective at dissociation of charge reduced CRP and ConA, SID showed better preserved subunit contacts that are useful for quaternary structure elucidation. In contrast, supercharging of CRP and ConA leads to facile dissociation into subunits even for CID. The extent of precursor unfolding also increases with greater charge. Another interesting finding is that low-charge multimer products (dimers, trimers, etc.) seem to be collapsed after being released from the complexes. Further investigation is necessary to fully understand this behavior. The data presented here suggest that charge manipulation can be used to "tune" the dissociation behavior of noncovalent protein complexes in order to obtain the most useful information desired for structural analysis.

A cluster-based chemical ionization method has been developed that produces protonated molecular ions from molecules introduced through a supersonic molecular beam interface. Mixed clusters of the analyte and a clustering agent (water or methanol) are produced in the expansion region of the beam, and are subsequently ionized by “fly through” electron impact (EI) ionization, which results in a mass spectrum that is a combination of protonated molecular ion peaks together with the conventional EI fragmentation pattern. The technique is presented and discussed as a tool complementary to electron impact ionization in supersonic molecular beams. Surface-induced dissociation on a rhenium oxide surface is also applied to simplify the mass spectra of clusters and reveal the analyte spectrum. The high gas flow rates involved with the supersonic molecular beam interface that enable the easy introduction of the clustering agents also have been used to introduce deuterating agents. An easy-to-use, fast, and routine on-line deuterium exchange method was developed to exchange active hydrogens (NH, OH). This method, combined with electron impact ionization, is demonstrated and discussed in terms of the unique information available through the EI fragmentation patterns, its ability to help in isomer identification, and possible applications with fast gas chromatography-mass spectrometry in supersonic molecular beams.

The electron impact mass spectrometry of straight chain alkanes C8H18-C40H82, squalane, methylstearate, 1-chlorohexadecane, 1-bromohexadecane, and dioctylphthalate was studied by sampling them with supersonic molecular beams. A fly-through Brink-type electron impact ion source was used, utilizing a vacuum background ion filtration technique based on differences between the kinetic energy of the supersonic beam species and that of thermal molecules. The 70-eV electron impact mass spectra of all the alkanes were characterized by a pronounced or dominant molecular weight peak together with all the fragment ions normally exhibited by the standard thermal 70-eV EI mass spectra. In contrast, the NIST library of most of these molecules did not show any molecular weight peak. By eliminating tile intramolecular thermal vibrational energy we gained control over the degree of molecular ion fragmentation by the electron energy. At an electron energy of 18 eV the molecular ion dissociation was further reduced considerably, with only a small absolute reduction in the peak height by less than a factor of 2. The effect of vibrational cooling increased with the molecular size and number of atoms. Pronounced differences were observed between the mass spectra of the straight chain triacontane and its branched isomer squalane. Similar mass spectra of octacosane (C28H58) achieved with 70-eV EI in a supersonic molecular beam were obtained with a magnetic sector mass spectrometer by using an electron energy of 14 eV and an ion source temperature of 150 °C. However, this ion source temperature precluded the gas chromatography-mass spectrometry (GC-MS) of octacosane. The GC-MS of alkanes was studied with an ion trap gas chromatograph-mass spectrometer at an ion source temperature of 230 °C. Thermal peak tailing was observed for C20H42 and heavier alkanes, whereas for C28H58 and heavier alkanes the severe peak tailing made quantitative GC-MS impractical. In contrast, no peak tailing existed even with C40H82 for GC-MS in supersonic molecular beams. The minimum detected amount of eicosane (C20, H42) was shown to be 60 fg. This was demonstrated by using single ion monitoring with the quadrupole mass analyzer tuned to the molecular weight peak of 282 u. The coupling of electron impact mass spectrometry in supersonic molecular beams with hyperthermal surface ionization and a fast GC-MS inlet is briefly discussed.

We report on the high-efficiency surface-induced dissociation of benzene and cyclohexane polyatomic ions after scattering from a rhenium oxide surface with a kinetic energy of 5-290 eV. Rhenium oxide was prepared by directly heating a rhenium metal foil, under 10−5 mbar partial oxygen pressure, at about 1000 K.Rhenium oxide is characterized by a very high work function of 6.4 eV and thus minimizes ion reneutralization probabilities. The catalytic combustion of surface organic impurities with oxygen ensures good long-term stability.We found that the surface-induced dissociation ion current is 70 times larger on rhenium oxide than on bare rhenium or stainless steel. Absolute scattered ion yields of about 50% were measured. The implications of surface-induced dissociation on mass spectrometry in supersonic molecular beams are mentioned.

A new approach for drug screening and detection in urine has been developed and studied. Fast gas chromatography/mass spectrometry (GC/MS) in combination with supersonic molecular beams was applied, employing hyperthermal surface ionization (HSI). Fast GC/MS (less than three minutes) screening of drugs in an untreated urine sample, at a detection limit of less than 100 ppb is shown. For this purpose, a new technique of “in-needle vaporization injection” (INVI) was developed for the introduction of untreated urine into the GC injector, leading to a minimal amount of salt and residue deposit on the GC liner and column. This new technique is compared with conventional syringe injection. Hyperthermal surface ionization of drugs is discussed in terms of ionization efficiency, selectivity and mass spectral appearance.