Characterization of 2-Butanone Peroxide Oligomeric Profiles and Their Associated Gas-Phase and Solution-Phase Rearrangement Products by Electrospray Ionization Mass Spectrometry for Forensic Applications.

2-Butanone peroxide (also known as methyl ethyl ketone peroxide, MEKP) has applications as a cross-linker in the chemical industry and is also encountered as a homemade primary high explosive; therefore, it is of interest to both process chemists and forensic examiners. Specifically for forensic applications, we demonstrate that when traditional synthetic procedures, available to any hobbyist, are utilized to generate MEKP, oligomeric peroxide units (n ≤ 12), along with several other oligomeric byproduct distributions, are readily observed by liquid chromatography-mass spectrometry (LC-MS). These oligomeric byproducts correspond to the formation of methyl/ethyl ketone end group(s) at the oligomer end group (i.e., loss of ethanol(s) and/or methanol(s) from the oligomer termini). Based on the interpretation of the MS and MS/MS behavior along with the characterization of newly generated terminal alkyl ketone products, we propose that these byproducts are consistent with a Hock-like rearrangement of the primary MEKP distribution in the acidified reaction medium. Following a procedure for homemade preparation, triplicate lots were synthesized. Unique oligomeric and byproduct distributions provided discriminatory power between the synthetic lots. Furthermore, the distributions of MEKP oligomers and the various byproducts in the initiated MEKP match the intensity distributions observed in the intact material with remarkable accuracy. This observation suggests that the postinitiation residue of MEKP could be associated or dissociated from a separately collected intact material obtained during an investigation by examining these oligomeric and byproduct profiles.

Online Bipolar Dual Spray for the Charge State Reduction and Characterization of Complex Synthetic Polymers.

Charge reduction mass spectrometry (CR/MS) hyphenated to liquid chromatography (LC) couples liquid-phase compound separation and mass spectral decompression to resolve and characterize multicomponent systems. LC/CR/MS has proven to be effective for complex mixture analysis, particularly synthetic polymers. A newer charge manipulation approach called bipolar dual spray has previously been demonstrated to reduce the observed charge state distribution of ammoniated polyethene glycol. In this approach, two electrospray emitters, in close proximity and of opposite polarity, fuse droplets from their electrospray plumes, which allows the subsequent chemistry. In this work, we investigate the ability of bipolar dual spray to reduce the charge of synthetic polyols, thereby simplifying complex mixture analysis and generating new compositional information only available through the coupling of charge reduction with LC/MS analysis. This work also represents the first demonstration of online charge reduction via dual spray. Polyethylene glycol (PEG) 7.2K subjected to LC/MS with dual spray reduced the average charge state from 8.2+ to 4.4+. LC/MS with dual spray was also applied to the characterization of an end-group-modified PEG 10K (i.e., aminated) containing several reaction impurities. This approach allowed for the identification of low-level starting material, tosylated PEG, and PEG mono(amine), where both LC/MS and direct infusion dual spray did not detect the impurities. Overall, the results demonstrated that bipolar dual spray can be incorporated into an LC/MS analysis and affords the ability to reduce the charge state distribution of PEG cations, decompress the m/z axis, lower spectra complexity, and enable/simplify data interpretation.

Stability Assessment of a Polymeric Brominated Flame Retardant in Polystyrene Foams under Application-Relevant Conditions.

The flame retardant (FR) BLUEDGE polymeric flame retardant (PFR) has been in use since 2011 and was developed as a replacement FR for hexabromocyclododecane in polystyrene (PS)-based insulation foams. To better understand the degradation behavior of the PFR used within PS foams, we examined the degradation of PFR under application-relevant conditions. Thermo-oxidative and photolytic pathways represent the most relevant degradation pathways. Separately, both the thermal and oxidative degradations of PFR at ambient conditions were shown to be negligible based on kinetic models of thermogravimetric analysis data obtained at elevated temperatures; the models predict that it would take 100 years to degrade 1% of PFR at 50 °C and 1000 years at 20 °C. Photodegradation was shown to degrade PFR after accelerated ultraviolet (UV) aging/exposure. UV radiation did not significantly penetrate the foam insulation (<2000 µm); the degradation process took place primarily at the surface. The molecular weight of the polymer changed with degradation, but there was minimal loss of bromine from the foam with degradation. The data from the liquid chromatography-mass spectrometry analysis focused primarily on several small-molecule polar products formed, which included two brominated species. These species were predicted using computer-based modeling to be biodegradable, to not be persistent in the environment, and to exhibit a low toxicity to aquatic organisms.

Microdroplet Fusion Chemistry for Charge State Reduction of Synthetic Polymers via Bipolar Dual Spray with Anionic Reagents.

Microdroplet fusion chemistry is an emerging area of analyte manipulation that utilizes the ion source region of a mass spectrometer to covalently derivatize or manipulate the charge state distribution. This technique utilizes two electrospray emitters in close proximity, where the droplets from each electrospray plume fuse and undergo the subsequent chemistry. In this study, microdroplet fusion chemistry via bipolar dual spray has demonstrated the ability to reduce the average charge state of polyethylene glycol (PEG) cations using anionic reagents. Bipolar dual spray was implemented on a commercial mass spectrometer with limited hardware modifications to the ion source. Reagents including ammonium hydroxide, formic acid, and lithium chloride showed dramatic shifts in the average charge state of PEG 3.8 K cations (e.g., 5.0+ to 2.5+) along with the emergence of newly detected charge states. An organic base, tributylamine, had no effect on the charge state distribution of PEG 3.8 K cations. These results were consistent with an ion-pairing mechanism, where reagent anions destabilized ammonium cation interactions with PEG 3.8 K upon droplet fusion from the negative and positive emitters. Additional bipolar dual spray experiments with PEG 12.6 K demonstrated the ability to transform uninterpretable mass information into distinct charge states ranging from [M+8NH4]+ to [M+3NH4]+. Overall, this study provides insight into the nature of dual spray chemistry and will aid future experimental design in microdroplet covalent chemistry.

Coupling Charge Reduction Mass Spectrometry to Liquid Chromatography for Complex Mixture Analysis.

Electrospray ionization (ESI) of solution mixtures often generates complex mass spectra, even following liquid chromatography (LC), due to analyte multiple charging. Multiple charge state distributions can lead to isobaric interferences, mass spectral congestion, and ambiguous ion identification. As a consequence, data interpretation increases in complexity. Several charge reduction mass spectrometry (MS) approaches have been previously developed to reduce the average charge state of gaseous ions; however, all of these techniques have been restricted to direct infusion MS. In this study, synthetic polyols and surfactants separated by liquid chromatography and ionized by positive mode ESI have been subjected to polonium-210 α-particle radiation to reduce the average charge state to singly charged cations prior to mass analysis. LC/MS analysis of 5000 molecular weight poly(ethylene glycol) (PEG5000) generated an average charge state of 5.88+; whereupon, liquid chromatography/electrospray ionization/charge reduction/mass spectrometry (LC/CR/MS) analysis of PEG 5000 generated an average charge state of 1.00+. The PEG5000 results demonstrated a decrease in spectral complexity and enabled facile interpretation. Other complex solution mixtures representing specific MS challenges (i.e., competitive ionization and isobaric ion overlap) were explored and analyzed with LC/CR/MS to demonstrate the benefits of coupling LC to CR/MS. For example, polyol information related to initiator, identity/relative amount of monomer, and estimated molecular weight was characterized in random and triblock ethylene oxide/propylene oxide polyols using LC/CR/MS. LC/CR/MS is a new analytical technique for the analysis of complex mixtures.

Gas-Phase Chemical Separation of Phosphatidylcholine and Phosphatidylethanolamine Cations via Charge Inversion Ion/Ion Chemistry.

The [M + H](+) cations formed upon electrospray ionization of the glycerophospholipids phosphatidylcholine (PC) and phosphatidylethanolamine (PE) show distinct reactivities upon gas-phase reactions with doubly deprotonated 1,4-phenylenedipropionic acid (PDPA). PC cations undergo charge inversion via adduct formation with subsequent methyl cation and proton transfer to the acid to yield [PC - CH3](-) anions. These demethylated PC anions fragment upon ion trap collision-induced dissociation (CID) to yield products that reveal fatty acid chain lengths and degrees of unsaturation. PE cations, on the other hand, undergo charge inversion via double proton transfer to the two carboxylate moieties in doubly deprotonated PDPA to yield [PE - H](-) anions. These anions also fragment upon ion trap CID to yield product ions indicative of chain lengths and degrees of unsaturation in the fatty acyl moieties. Advantage is taken of this distinct reactivity to separate isomeric and isobaric PC and PE cations present in mass spectra of lipid mixtures. A cation precursor ion population containing a mixture of PE and PC cations is mass-selected and subjected to ion/ion charge inversion reactions that result in separation of PC and PE anions into different mass-to-charge ratios. Mass selection and subsequent ion trap CID of the lipid anions allows for the characterization of the isomeric lipids within each subclass. The charge inversion approach described here is demonstrated to provide increased signal-to-noise ratios for detection of PCs and PEs relative to the standard negative ionization approach as well as improved mixture analysis performance.

Strategies for the Gas Phase Modification of Cationized Arginine via Ion/ion Reactions.

The gas phase acetylation of cationized arginine residues is demonstrated here using ion/ion reactions with sulfosuccinimidyl acetate (sulfo-NHS acetate) anions. Previous reports have demonstrated the gas phase modification of uncharged primary amine (the N-terminus and ε-amino side chain of lysine) and uncharged guanidine (the arginine side chain) functionalities via sulfo-NHS ester chemistry. Herein, charge-saturated arginine-containing peptides that contain sodium ions as the charge carriers, such as [ac-ARAAARA+2Na]2+, are shown to exhibit strong reactivity towards sulfo-NHS acetate whereas the protonated peptide analogues exhibit no such reactivity. This difference in reactivity is attributed to the lower sodium ion (as compared to proton) affinity of the arginine, which results in increased nucleophilicity of the cationized arginine guanidinium functionality. This increased nucleophilicity improves the arginine residue's reactivity towards sulfo-NHS esters and enhances the gas phase covalent modification pathway. No such dramatic increase in reactivity towards sulfo-NHS acetate has been observed upon sodium cationization of lysine amino acid residues, indicating that this behavior appears to be unique to arginine. The sodium cationization process is demonstrated in the condensed phase by simply spiking sodium chloride into the peptide sample solution and in the gas phase by a peptide-sodium cation exchange process with a sulfo-NHS acetate sodium-bound dimer cluster reagent. This methodology demonstrates several ways by which arginine can be covalently modified in the gas phase even when it is charged. Collisional activation of an acetylated arginine product can result in deguanidination of the residue, generating an ornithine. This gas phase ornithination exhibits similar site-specific fragmentation behavior to that observed with peptides ornithinated in solution and may represent a useful approach for inducing selective peptide cleavages.

Reagent cluster anions for multiple gas-phase covalent modifications of peptide and protein cations.

Multiple gas phase ion/ion covalent modifications of peptide and protein ions are demonstrated using cluster-type reagent anions of N-hydroxysulfosuccinimide acetate (sulfo-NHS acetate) and 2-formyl-benzenesulfonic acid (FBMSA). These reagents are used to selectively modify unprotonated primary amine functionalities of peptides and proteins. Multiple reactive reagent molecules can be present in a single cluster ion, which allows for multiple covalent modifications to be achieved in a single ion/ion encounter and at the 'cost' of only a single analyte charge. Multiple derivatizations are demonstrated when the number of available reactive sites on the analyte cation exceeds the number of reagent molecules in the anionic cluster (e.g., data shown here for reactions between the polypeptide [K10 + 3H](3+) and the reagent cluster [5R(5Na) - Na](-)). This type of gas-phase ion chemistry is also applicable to whole protein ions. Here, ubiquitin was successfully modified using an FBMSA cluster anion which, upon collisional activation, produced fragment ions with various numbers of modifications. Data for the pentamer cluster are included as illustrative of the results obtained for the clusters comprised of two to six reagent molecules.

Gas-phase transformation of phosphatidylcholine cations to structurally informative anions via ion/ion chemistry.

Gas-phase transformation of synthetic phosphatidylcholine (PC) monocations to structurally informative anions is demonstrated via ion/ion reactions with doubly deprotonated 1,4-phenylenedipropionic acid (PDPA). Two synthetic PC isomers, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (PC(16:0/18:1)) and 1-oleoyl-2-palmitoyl-sn-glycero-3-phosphocholine (PC(18:1/16:0)), were subjected to this ion/ion chemistry. The product of the ion/ion reaction is a negatively charged complex, [PC + PDPA - H](-). Collisional activation of the long-lived complex causes transfer of a proton and methyl cation to PDPA, generating [PC - CH3](-). Subsequent collisional activation of the demethylated PC anions produces abundant fatty acid carboxylate anions and low-abundance acyl neutral losses as free acids and ketenes. Product ion spectra of [PC - CH3](-) suggest favorable cleavage at the sn-2 position over the sn-1 due to distinct differences in the relative abundances. In contrast, collisional activation of PC cations is absent of abundant fatty acid chain-related product ions and typically indicates only the lipid class via formation of the phosphocholine cation. A solution phase method to produce the gas-phase adducted PC anion is also demonstrated. Product ion spectra derived from the solution phase method are similar to the results generated via ion/ion chemistry. This work demonstrates a gas-phase means to increase structural characterization of phosphatidylcholines via ion/ion chemistry.

Gas-phase reactivity of carboxylic acid functional groups with carbodiimides.

Gas-phase modification of carboxylic acid functionalities is performed via ion/ion reactions with carbodiimide reagents [N-cyclohexyl-N'-(2-morpholinoethyl)carbodiimide (CMC) and [3-(3-Ethylcarbodiimide-1-yl)propyl]trimethylaminium (ECPT)]. Gas-phase ion/ion covalent chemistry requires the formation of a long-lived complex. In this instance, the complex is stabilized by an electrostatic interaction between the fixed charge quaternary ammonium group of the carbodiimide reagent cation and the analyte dianion. Subsequent activation results in characteristic loss of an isocyanate derivative from one side of the carbodiimide functionality, a signature for this covalent chemistry. The resulting amide bond is formed on the analyte at the site of the original carboxylic acid. Reactions involving analytes that do not contain available carboxylic acid groups (e.g., they have been converted to sodium salts) or reagents that do not have the carbodiimide functionality do not undergo a covalent reaction. This chemistry is demonstrated using PAMAM generation 0.5 dendrimer, ethylenediaminetetraacetic acid (EDTA), and the model peptide DGAILDGAILD. This work demonstrates the selective gas-phase covalent modification of carboxylic acid functionalities.

Ion/ion reactions of MALDI-derived peptide ions: increased sequence coverage via covalent and electrostatic modification upon charge inversion.

Atmospheric pressure matrix-assisted laser desorption/ionization (AP-MALDI)-derived tryptic peptide ions have been subjected to ion/ion reactions with doubly deprotonated 4-formyl-1,3-benzenedisulfonic acid (FBDSA) in the gas-phase. The ion/ion reaction produces a negatively charged electrostatic complex composed of the peptide cation and reagent dianion, whereupon dehydration of the complex via collision-induced dissociation (CID) produces a Schiff base product anion. Collisional activation of modified lysine-terminated tryptic peptide anions is consistent with a covalent modification of unprotonated primary amines (i.e., N-terminus and ε-NH(2) of lysine). Modified arginine-terminated tryptic peptides have shown evidence of a covalent modification at the N-terminus and a noncovalent interaction with the arginine residue. The modified anions yield at least as much sequence information upon CID as the unmodified cations for the small tryptic peptides examined here and more sequence information for the large tryptic peptides. This study represents the first demonstration of gas-phase ion/ion reactions involving MALDI-derived ions. In this case, covalent and electrostatic modification charge inversion is shown to enhance MALDI tandem mass spectrometry of tryptic peptides.

Covalent and non-covalent binding in the ion/ion charge inversion of peptide cations with benzene-disulfonic acid anions.

Protonated angiotensin II and protonated leucine enkephalin-based peptides, which included YGGFL, YGGFLF, YGGFLH, YGGFLK and YGGFLR, were subjected to ion/ion reactions with the doubly deprotonated reagents 4-formyl-1,3-benzenedisulfonic acid (FBDSA) and 1,3-benzenedisulfonic acid (BDSA). The major product of the ion/ion reaction is a negatively charged complex of the peptide and reagent. Following dehydration of [M + FBDSA-H](-) via collisional-induced dissociation (CID), angiotensin II (DRVYIHPF) showed evidence for two product populations, one in which a covalent modification has taken place and one in which an electrostatic modification has occurred (i.e. no covalent bond formation). A series of studies with model systems confirmed that strong non-covalent binding of the FBDSA reagent can occur with subsequent ion trap CID resulting in dehydration unrelated to the adduct. Ion trap CID of the dehydration product can result in cleavage of amide bonds in competition with loss of the FBDSA adduct. This scenario is most likely for electrostatically bound complexes in which the peptide contains both an arginine residue and one or more carboxyl groups. Otherwise, loss of the reagent species from the complex, either as an anion or as a neutral species, is the dominant process for electrostatically bound complexes. The results reported here shed new light on the nature of non-covalent interactions in gas phase complexes of peptide ions that can be used in the rationale design of reagent ions for specific ion/ion reaction applications.

Dissociation Behavior of Tryptic and Intramolecular Disulfide-linked Peptide Ions Modified in the Gas Phase via Ion/Ion Reactions.

Protonated tryptic peptides, somatostatin-14, and oxytocin have been subjected to reactions with doubly deprotonated 4-formyl-1,3-benzenedisulfonic acid (FBDSA) in the gas phase. The major product is a negatively-charged complex comprised of the peptide and the reagent. Upon dehydration of the complex, all peptides show evidence for Schiff base formation involving a primary amine of the peptide. Some peptides also show evidence for the formation of a relatively strong electrostatic interaction without Schiff base formation (i.e., a mixture of isomeric precursor ions is generated upon dehydration of the complex). Ion trap collision-induced dissociation of the dehydration products from all peptides examined gave distinct product ion spectra relative to the deprotonated and protonated forms of the peptides. The distinct behavior of the modified ions is attributed to the highly stable charge carrying sulfonate group, which tends to inhibit intramolecular proton transfer in negatively charged species. Modified anions of the peptides with an intramolecular disulfide linkage show evidence for cleavage of both the disulfide linkage and an amide bond in the loop defined by the disulfide bond. Modification of protonated peptides via charge inversion with FBDSA is a useful means for generating novel and distinct ion-types that can provide complementary structural information upon subsequent activation to that obtained from dissociation of protonated or deprotonated forms of the peptide.

Gas-phase bioconjugation of peptides via ion/ion charge inversion: Schiff base formation on the conversion of cations to anions.

The selective covalent modification of singly protonated peptides in the gas-phase via ion/ion charge inversion reactions is demonstrated. Doubly deprotonated 4-formyl-1,3-benzene disulfonic acid serves as a reagent anion for forming a Schiff base via the reaction of a primary amine on the peptide and the aldehyde functionality of the reagent anion. The process is initiated by the formation of an ion/ion complex comprised of the two reactants. Ion trap collisional activation of the complex results in loss of water from the intermediate that gives rise to Schiff base formation. N-terminally acetylated peptides with no lysine residues do not undergo covalent bond formation upon reaction with the reagent anion. Rather, the adduct species simply loses the reagent either as a neutral species or as a deprotonated species. The ability to modify singly protonated peptide ions covalently and selectively opens up new possibilities for the analysis of peptides and, possibly, other analyte species with primary amine functionalities.