Improved sampling and detection of ignitable liquid residues and explosives by mass spectrometry and ion mobility spectrometry

Fire debris evidence is submitted to crime laboratories to determine if an ignitable liquid (IL) accelerant was used to commit arson. An ignitable liquid residue (ILR) may be difficult to analyze due to interferences, complex matrices, degradation, and low concentrations of analytes. Debris from an explosion and pre-detonated explosive compounds are not trivial to detect and identify due to sampling difficulties, complex matrices, and extremely low amounts (nanogram) of material present. The focus of this research is improving the sampling and detection of ILR and explosives through enhanced sensitivity, selectivity, and field portable instrumentation. Solid Phase MicroExtraction (SPME) enhanced the extraction of ILR by two orders of magnitude over conventional activated charcoal strip (ACS) extraction. Gas chromatography tandem mass spectrometry (GC/MS/MS) improved sensitivity of ILR by one order of magnitude and explosives by two orders of magnitude compared to gas chromatography mass spectrometry (GC/MS). Improvements in sensitivity were attributed to enhanced selectivity. An interface joining SPME to ion mobility spectrometry (IMS) has been constructed and evaluated to improve field detection of hidden explosives. The SPME-IMS interface improved the detection of volatile and semi-volatile explosive compounds and successfully adapted the IMS from a particle sampler into a vapor sampler. ^

Assessment of the occurrence and potential risks of pharmaceuticals and their metabolites in fish and water using liquid chromatography mass spectrometry

A comprehensive method for the analysis of 11 target pharmaceuticals representing multiple therapeutic classes was developed for biological tissues (fish) and water. Water samples were extracted using solid phase extraction (SPE), while fish tissue homogenates were extracted using accelerated solvent extraction (ASE) followed by mixed-mode cation exchange SPE cleanup and analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS). Among the 11 target pharmaceuticals analyzed, trimethoprim, caffeine, sulfamethoxazole, diphenhydramine, diltiazem, carbamazepine, erythromycin and fluoxetine were consistently detected in reclaimed water. On the other hand, caffeine, diphenhydramine and carbamazepine were consistently detected in fish and surface water samples. In order to understand the uptake and depuration of pharmaceuticals as well as bioconcentration factors (BCFs) under the worst-case conditions, mosquito fish were exposed to reclaimed water under static-renewal for 7 days, followed by a 14-day depuration phase in clean water. Characterization of the exposure media revealed the presence of 26 pharmaceuticals while 5 pharmaceuticals including caffeine, diphenhydramine, diltiazem, carbamazepine, and ibuprofen were present in the organisms as early as 5 h from the start of the exposure. Liquid chromatography ultra-high resolution Orbitrap mass spectrometry was explored as a tool to identify and quantify phase II pharmaceutical metabolites in reclaimed water. The resulting data confirmed the presence of acetyl-sulfamethoxazole and sulfamethoxazole glucuronide in reclaimed water. To my knowledge, this is the first known report of sulfamethoxazole glucuronide surviving intact through wastewater treatment plants and occurring in environmental water samples. Finally, five bioaccumulative pharmaceuticals including caffeine, carbamazepine, diltiazem, diphenhydramine and ibuprofen detected in reclaimed water were investigated regarding the acute and chronic risks to aquatic organisms. The results indicated a low potential risk of carbamazepine even under the worst case exposure scenario. Given the dilution factors that affect environmental releases, the risk of exposure to carbamazepine will be even more reduced.

High resolution mass spectrometry for quantitative analysis and untargeted screening of algal toxins in mussels and passive samplers

Measurement of marine algal toxins has traditionally focussed on shellfish monitoring while, over the last decade, passive sampling has been introduced as a complementary tool for exploratory studies. Since 2011, liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been adopted as the EU reference method (No.15/2011) for detection and quantitation of lipophilic toxins. Traditional LC-MS approaches have been based on low-resolution mass spectrometry (LRMS), however, advances in instrument platforms have led to a heightened interest in the use of high-resolution mass spectrometry (HRMS) for toxin detection. This work describes the use of HRMS in combination with passive sampling as a progressive approach to marine algal toxin surveys. Experiments focused on comparison of LRMS and HRMS for determination of a broad range of toxins in shellfish and passive samplers. Matrix effects are an important issue to address in LC-MS; therefore, this phenomenon was evaluated for mussels (Mytilus galloprovincialis) and passive samplers using LRMS (triple quadrupole) and HRMS (quadrupole time-of-flight and Orbitrap) instruments. Matrix-matched calibration solutions containing okadaic acid and dinophysistoxins, pectenotoxin, azaspiracids, yessotoxins, domoic acid, pinnatoxins, gymnodimine A and 13-desmethyl spirolide C were prepared. Similar matrix effects were observed on all instruments types. Most notably, there was ion enhancement for pectenotoxins, okadaic acid/dinophysistoxins on one hand, and ion suppression for yessotoxins on the other. Interestingly, the ion selected for quantitation of PTX2 also influenced the magnitude of matrix effects, with the sodium adduct typically exhibiting less susceptibility to matrix effects than the ammonium adduct. As expected, mussel as a biological matrix, quantitatively produced significantly more matrix effects than passive sampler extracts, irrespective of toxin. Sample dilution was demonstrated as an effective measure to reduce matrix effects for all compounds, and was found to be particularly useful for the non-targeted approach. Limits of detection and method accuracy were comparable between the systems tested, demonstrating the applicability of HRMS as an effective tool for screening and quantitative analysis. HRMS offers the advantage of untargeted analysis, meaning that datasets can be retrospectively analysed. HRMS (full scan) chromatograms of passive samplers yielded significantly less complex data sets than mussels, and were thus more easily screened for unknowns. Consequently, we recommend the use of HRMS in combination with passive sampling for studies investigating emerging or hitherto uncharacterised toxins.

Assessment of the Occurrence and Potential Risks of Pharmaceuticals and their Metabolites in Fish and Water Using Liquid Chromatography Mass Spectrometry

A comprehensive method for the analysis of 11 target pharmaceuticals representing multiple therapeutic classes was developed for biological tissues (fish) and water. Water samples were extracted using solid phase extraction (SPE), while fish tissue homogenates were extracted using accelerated solvent extraction (ASE) followed by mixed-mode cation exchange SPE cleanup and analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS). Among the 11 target pharmaceuticals analyzed, trimethoprim, caffeine, sulfamethoxazole, diphenhydramine, diltiazem, carbamazepine, erythromycin and fluoxetine were consistently detected in reclaimed water. On the other hand, caffeine, diphenhydramine and carbamazepine were consistently detected in fish and surface water samples. In order to understand the uptake and depuration of pharmaceuticals as well as bioconcentration factors (BCFs) under the worst-case conditions, mosquito fish were exposed to reclaimed water under static-renewal for 7 days, followed by a 14-day depuration phase in clean water. Characterization of the exposure media revealed the presence of 26 pharmaceuticals while 5 pharmaceuticals including caffeine, diphenhydramine, diltiazem, carbamazepine, and ibuprofen were present in the organisms as early as 5 h from the start of the exposure. Liquid chromatography ultra-high resolution Orbitrap mass spectrometry was explored as a tool to identify and quantify phase II pharmaceutical metabolites in reclaimed water. The resulting data confirmed the presence of acetyl-sulfamethoxazole and sulfamethoxazole glucuronide in reclaimed water. To my knowledge, this is the first known report of sulfamethoxazole glucuronide surviving intact through wastewater treatment plants and occurring in environmental water samples. Finally, five bioaccumulative pharmaceuticals including caffeine, carbamazepine, diltiazem, diphenhydramine and ibuprofen detected in reclaimed water were investigated regarding the acute and chronic risks to aquatic organisms. The results indicated a low potential risk of carbamazepine even under the worst case exposure scenario. Given the dilution factors that affect environmental releases, the risk of exposure to carbamazepine will be even more reduced.

Structure-inhibition relationship of phenylethanoid glycosides on angiotensin-converting enzyme using ultra-performance liquid chromatography-tandem quadrupole mass spectrometry

Angiotensin-converting enzyme (ACE) plays a critical role in rennin-angiotensin system. Recently, natural products isolated from herbal medicines revealed inhibitory effects against ACE which suggested their potential activities in regulating blood pressure. In this study, ACE inhibition (ACEI) of 21 phenylethanoid glycosides and related phenolic compounds were investigated by measuring the production of HA a rapid, sensitive, accurate and specific ultra-performance liquid chromatography-tandem quadrupole mass spectrometry (UPLC-MS/MS) method. The test compounds showed different inhibitory potencies on ACE ranging from 5.29 to 95.01% at 50 mM, and the compounds with ACEI higher than 50% were selected for further IC50 determination. The IC50 values were from 0.53 ± 0.04 to 15.035 ± 0.036 mM. The structure-inhibition relationship were then explored and the result showed that cinnamoyl groups played an essential role in ACEI of phenylethanoid glycosides. Furthermore, the sub-structures of increasing ACEI for phenylethanoid glycosides is more hydroxyls and less steric hindrance to chelate the active site Zn2+ of ACE. In summary, our results suggested that phenylethanoid glycosides are a widely available source of anti-hypertensive natural products and the information provided from structure-inhibition relationship study could aid the design of structurally modified phenylethanoid glycosides as anti-hypertensive drugs.

Automated de novo sequencing of proteins by tandem high-resolution mass spectrometry

A de novo sequencing program for proteins is described that uses tandem MS data from electron capture dissociation and collisionally activated dissociation of electrosprayed protein ions. Computer automation is used to convert the fragment ion mass values derived from these spectra into the most probable protein sequence, without distinguishing Leu/Ile. Minimum human input is necessary for the data reduction and interpretation. No extra chemistry is necessary to distinguish N- and C-terminal fragments in the mass spectra, as this is determined from the electron capture dissociation data. With parts-per-million mass accuracy (now available by using higher field Fourier transform MS instruments), the complete sequences of ubiquitin (8.6 kDa) and melittin (2.8 kDa) were predicted correctly by the program. The data available also provided 91% of the cytochrome c (12.4 kDa) sequence (essentially complete except for the tandem MS-resistant region K13–V20 that contains the cyclic heme). Uncorrected mass values from a 6-T instrument still gave 86% of the sequence for ubiquitin, except for distinguishing Gln/Lys. Extensive sequencing of larger proteins should be possible by applying the algorithm to pieces of ≈10-kDa size, such as products of limited proteolysis.

Elucidation of double bond position in unsaturated lipids by ozone electrospray ionization mass spectrometry

The position(s) of carbon-carbon double bonds within lipids can dramatically affect their structure and reactivity and thus has a direct bearing on biological function. Commonly employed mass spectrometric approaches to the characterization of complex lipids, however, fail to localize sites of unsaturation within the molecular structure and thus cannot distinguish naturally occurring regioisomers. In a recent communication \[Thomas, M. C.; Mitchell, T. W.; Blanksby, S. J. J. Am. Chem. Soc. 2006, 128, 58-59], we have presented a new technique for the elucidation of double bond position in glycerophospholipids using ozone-induced fragmentation within the source of a conventional electrospray ionization mass spectrometer. Here we report the on-line analysis, using ozone electrospray mass spectrometry (OzESI-MS), of a broad range of common unsaturated lipids including acidic and neutral glycerophospholipids, sphingomyelins, and triacylglycerols. All lipids analyzed are found to form a pair of chemically induced fragment ions diagnostic of the position of each double bond(s) regardless of the polarity, the number of charges, or the adduction (e.g., \[M - H](-), \[M - 2H](2-), \[M + H](+), \[M + Na](+), \[M + NH4](+)). The ability of OzESI-MS to distinguish lipids that differ only in the position of the double bonds is demonstrated using the glycerophosphocholine standards, GPCho(9Z-18:1/9Z-18:1) and GPCho(6Z-18:1/6Z-18:1). While these regioisomers cannot be differentiated by their conventional tandem mass spectra, the OzESI-MS spectra reveal abundant fragment ions of distinctive mass-to-charge ratio (m/z). The approach is found to be sufficiently robust to be used in conjunction with the m/z 184 precursor ion scans commonly employed for the identification of phosphocholine-containing lipids in shotgun lipidomic analyses. This tandem OzESI-MS approach was used, in conjunction with conventional tandem mass spectral analysis, for the structural characterization of an unknown sphingolipid in a crude lipid extract obtained from a human lens. The OzESI-MS data confirm the presence of two regioisomers, namely, SM(d18:0/15Z-24:1) and SM(d18:0/17Z-24:1), and suggest the possible presence of a third isomer, SM(d18:0/19Z-24:1), in lower abundance. The data presented herein demonstrate that OzESI-MS is a broadly applicable, on-line approach for structure determination and, when used in conjunction with established tandem mass spectrometric methods, can provide near complete structural characterization of a range of important lipid classes. As such, OzESI-MS may provide important new insight into the molecular diversity of naturally occurring lipids.

Investigation of heptakis(2,6-di-O-methyl)-beta-cyclodextrin inclusion complexes with flavonoid glycosides by electrospray ionization mass spectrometry

The non-covalent complexes between three flavonoid glycosides (quercitrin, hyperoside and rutin) and heptakis(2,6-di-O-methyl)-beta-cyclodextrin (DM-beta-CD) were investigated by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS). The 1:1 complexation of each flavonoid glycoside (guest) to the DM-beta-CD (host) was monitored in the negative ion mode by mixing each guest with an up to 30-fold molar excess of the host. The binding constants for all complexes were calculated by a linear equation in the order: DM-beta-CD:quercitrin > DM-beta-CD:rutin > DM-beta-CD:hyperoside. A binding model for the complexes has also been proposed based on the binding constants and tandem mass spectrometric data of these complexes.

A study of isomeric diglycosyl flavonoids by SORI CID of Fourier transform ion cyclotron mass Spectrometry in negative ion mode

High-resolution Sustained off resonance irradiation (SORI) CID was employed to distinguish four pairs of isomeric diglycosyl flavonoids in the negative mode using the electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI FTICR MS). All of these isomers can be distinguished via MS/MS data. For these diglycosyl flavones and flavanones, the deprotonated alpha 1-->6 linkage diglycosyl flavonoids produce fewer fragments than the alpha 1-->2 linkage type compounds and the Retro-Diels-Alder (RDA) reaction in MS/MS only takes place when the aglycone is a flavanone and glycosylated with an alpha 1-->2 intersaccharide linkage disaccharide. The deprotonation sites after collisional activation are discussed according to the high mass accuracy and high-resolution data of tandem spectrometry. Some of these high-resolution SORI CID product ions from alpha 1-->2 linkage diglycosyl flavonoids involve multibond cleavages; the possible mechanism is discussed based on the computer modeling using Gaussian 03 program package at the B3LYP/6-31G level of theory. Unambiguous elementary composition data provides fragmentation information that has not been reported previously.

Can density functional theory (DFT) be used as an aid to a deeper understanding of tandem mass spectrometric fragmentation pathways?

Prediction of tandem mass spectrometric (MS/MS) fragmentation for non-peptidic molecules based on structure is of immense interest to the mass spectrometrist. If a reliable approach to MS/MS prediction could be achieved its impact within the pharmaceutical industry could be immense. Many publications have stressed that the fragmentation of a molecular ion or protonated molecule is a complex process that depends on many parameters, making prediction difficult. Commercial prediction software relies on a collection of general heuristic rules of fragmentation, which involve cleaving every bond in the structure to produce a list of 'expected' masses which can be compared with the experimental data. These approaches do not take into account the thermodynamic or molecular orbital effects that impact on the molecule at the point of protonation which could influence the potential sites of bond cleavage based on the structural motif. A series of compounds have been studied by examining the experimentally derived high-resolution MS/MS data and comparing it with the in silico modelling of the neutral and protonated structures. The effect that protonation at specific sites can have on the bond lengths has also been determined. We have calculated the thermodynamically most stable protonated species and have observed how that information can help predict the cleavage site for that ion. The data have shown that this use of in silico techniques could be a possible way to predict MS/MS spectra. Copyright (C) 2009 John Wiley & Sons, Ltd.

Liquid AP-UV-MALDI enables stable ion yields of multiply charged peptide and protein ions for sensitive analysis by mass spectrometry

In biological mass spectrometry (MS), two ionization techniques are predominantly employed for the analysis of larger biomolecules, such as polypeptides. These are nano-electrospray ionization [1, 2] (nanoESI) and matrix-assisted laser desorption/ionization [3, 4] (MALDI). Both techniques are considered to be “soft”, allowing the desorption and ionization of intact molecular analyte species and thus their successful mass-spectrometric analysis. One of the main differences between these two ionization techniques lies in their ability to produce multiply charged ions. MALDI typically generates singly charged peptide ions whereas nanoESI easily provides multiply charged ions, even for peptides as low as 1000 Da in mass. The production of highly charged ions is desirable as this allows the use of mass analyzers, such as ion traps (including orbitraps) and hybrid quadrupole instruments, which typically offer only a limited m/z range (< 2000–4000). It also enables more informative fragmentation spectra using techniques such as collisioninduced dissociation (CID) and electron capture/transfer dissociation (ECD/ETD) in combination with tandem MS (MS/MS). [5, 6] Thus, there is a clear advantage of using ESI in research areas where peptide sequencing, or in general, the structural elucidation of biomolecules by MS/MS is required. Nonetheless, MALDI with its higher tolerance to contaminants and additives, ease-of-operation, potential for highspeed and automated sample preparation and analysis as well as its MS imaging capabilities makes it an ionization technique that can cover bioanalytical areas for which ESI is less suitable. [7, 8] If these strengths could be combined with the analytical power of multiply charged ions, new instrumental configurations and large-scale proteomic analyses based on MALDI MS(/MS) would become feasible.

Separating the wheat from the chaff: unbiased filtering of background tandem mass spectra improves protein identification

Only a small fraction of spectra acquired in LC-MS/MS runs matches peptides from target proteins upon database searches. The remaining, operationally termed background, spectra originate from a variety of poorly controlled sources and affect the throughput and confidence of database searches. Here, we report an algorithm and its software implementation that rapidly removes background spectra, regardless of their precise origin. The method estimates the dissimilarity distance between screened MS/MS spectra and unannotated spectra from a partially redundant background library compiled from several control and blank runs. Filtering MS/MS queries enhanced the protein identification capacity when searches lacked spectrum to sequence matching specificity. In sequence-similarity searches it reduced by, on average, 30-fold the number of orphan hits, which were not explicitly related to background protein contaminants and required manual validation. Removing high quality background MS/MS spectra, while preserving in the data set the genuine spectra from target proteins, decreased the false positive rate of stringent database searches and improved the identification of low-abundance proteins.

RAMSY: Ratio Analysis of Mass Spectrometry to Improve Compound Identification

The complexity of biological samples poses a major challenge for reliable compound identification in mass spectrometry (MS). The presence of interfering compounds that cause additional peaks in the spectrum can make interpretation and assignment difficult. To overcome this issue, new approaches are needed to reduce complexity and simplify spectral interpretation. Recently, focused on unknown metabolite identification, we presented a new approach, RANSY (ratio analysis of nuclear magnetic resonance spectroscopy; Anal. Chem. 2011, 83, 7616-7623), which extracts the signals related to the same metabolite based on peak intensity ratios. On the basis of this concept, we present the ratio analysis of mass spectrometry (RAMSY) method, which facilitates improved compound identification in complex MS spectra. RAMSY works on the principle that, under a given set of experimental conditions, the abundance/intensity ratios between the mass fragments from the same metabolite are relatively constant. Therefore, the quotients of average peak ratios and their standard deviations, generated using a small set of MS spectra from the same ion chromatogram, efficiently allow the statistical recovery of the metabolite peaks and facilitate reliable identification. RAMSY was applied to both gas chromatography/MS and liquid chromatography tandem MS (LC-MS/MS) data to demonstrate its utility. The performance of RAMSY is typically better than the results from correlation methods. RAMSY promises to improve unknown metabolite identification for MS users in metabolomics or other fields.

Gas-phase reactivity of protonated 2-oxazoline derivatives: mass spectrometry and computational studies

RATIONALE: Oxazolines have attracted the attention of researchers worldwide due to their versatility as carboxylic acid protecting groups, chiral auxiliaries, and ligands for asymmetric catalysis. Electrospray ionization tandem mass spectrometric (ESI-MS/MS) analysis of five 2-oxazoline derivatives has been conducted, in order to understand the influence of the side chain on the gas-phase dissociation of these protonated compounds under collision-induced dissociation (CID) conditions. METHODS: Mass spectrometric analyses were conducted in a quadrupole time-of-flight (Q-TOF) spectrometer fitted with electrospray ionization source. Protonation sites have been proposed on the basis of the gas-phase basicity, proton affinity, atomic charges, and a molecular electrostatic potential map obtained on the basis of the quantum chemistry calculations at the B3LYP/6-31 + G(d, p) and G2(MP2) levels. RESULTS: Analysis of the atomic charges, gas-phase basicity and proton affinities values indicates that the nitrogen atom is a possible proton acceptor site. On the basis of these results, two main fragmentation processes have been suggested: one taking place via neutral elimination of the oxazoline moiety (99 u) and another occurring by sequential elimination of neutral fragments with 72 u and 27 u. These processes should lead to formation of R+. CONCLUSIONS: The ESI-MS/MS experiments have shown that the side chain could affect the dissociation mechanism of protonated 2-oxazoline derivatives. For the compound that exhibits a hydroxyl at the lateral chain, water loss has been suggested to happen through an E2-type elimination, in an exothermic step. Copyright (C) 2012 John Wiley & Sons, Ltd.