Development of in vitro methods to model the impact of vaginal lactobacilli on Staphylococcus aureus biofilm formation on menstrual cups as well as validation of recommended cleaning directions.

Introduction: Menstrual cups (MC) are a reusable feminine hygiene product. A recent publication suggested that Staphylococcus aureus (S. aureus) biofilms can form on MCs which may lead to increased risk of menstrual Toxic Shock Syndrome (mTSS). Additionally, there is concern that buildup of residual menses may contribute to microbial growth and biofilm formation further increasing mTSS risk. Quantitative and qualitative analysis of in vitro tests were utilized to determine if S. aureus biofilm could form on MC in the presence of the keystone species Lactobacillus after 12 h of incubation. The methodology was based on a modification of an anaerobic in vitro method that harnesses the keystone species hypothesis by including a representative of vaginal lactic acid bacteria. Methods: MCs were incubated anaerobically for 12 h in Vaginal Defined Media (VDM) with the two morphologically distinct bacteria, Lactobacillus gasseri (L. gasseri) and S. aureus. Colony Forming Units (CFU) for each organism from the VDM broth and sonicated MC were estimated. In addition, a separate experiment was conducted where S. aureus was grown for 12 h in the absence of L. gasseri. Qualitative analysis for biofilm formation utilized micro-CT (µ-CT) and cryogenic scanning electron microscopy (Cryo-SEM). Results: Samples collected from the media control had expected growth of both organisms after 12 h of incubation. Samples collected from VDM broth were similar to media control at the end of the 12-h study. Total S. aureus cell density on MC following sonication/rinsing was minimal. Results when using a monoculture of S. aureus demonstrated that there was a significant growth of the organism in the media control and broth as well as the sonicated cups indicating that the presence of L. gasseri was important for controlling growth and adherence of S. aureus. Few rod-shaped bacteria (L. gasseri) and cocci (S. aureus) could be identified on the MCs when grown in a dual species culture inoculum and no biofilm was noted via µ-CT and cryo-SEM. Additionally, efforts to model and understand the validity of the current labeled recommendations for MC cleaning in-between uses are supported. Discussion: The data support continued safe use of the Tampax® cup when used and maintained as recommended.

Characterization of ionized lignin model compounds with α-O-4 linkages by positive- and negative-ion mode electrospray ionization tandem mass spectrometry based on collision-activated dissociation.

RATIONALE: The biggest obstacle in the rational conversion of biomass into aromatic chemicals is the identification of unknown compounds in lignin degradation mixtures that are highly complex. As opposed to lignin degradation products with ß-O-4 linkages, very little is known about the mass spectrometric analysis of lignin degradation products with α-O-4 linkages. METHODS: Lignin model compounds with an α-O-4 and another linkage, as well as lignin model compounds with only ß-O-4 linkages, were ionized by attachment of lithium or sodium cations under positive-ion mode electrospray ionization (ESI) or by deprotonation in negative-ion mode ESI in a linear quadrupole ion trap mass spectrometer. The ions were subjected to collision-activated dissociation in multiple-stage tandem mass spectrometry experiments to characterize their fragmentation patterns. RESULTS: All studied compounds formed abundant sodium and lithium cation adducts in positive-ion mode ESI with no fragmentation. Model compounds with ß-O-4 linkages displayed stable [M - H]- ions in negative-ion mode ESI whereas compounds with α-O-4 linkages only showed fragment ions. CAD of the lithiated α-O-4 compounds provided more structural information than CAD of sodiated compounds. However, both sodiated and lithiated compounds with α-O-4 linkages showed losses of monomer units at the MS2 stage, which is useful for sequencing of lignins with this type of linkage. CONCLUSIONS: An ionization and sequencing method has been developed for lignin model compounds with α-O-4 linkages that spontaneously fragment upon ionization via (-)ESI.

Quinoline Triradicals: A Reactivity Study.

The gas-phase reactivities of several protonated quinoline-based σ-type (carbon-centered) mono-, bi-, and triradicals toward dimethyl disulfide (DMDS) were studied by using a linear quadrupole ion trap mass spectrometer. The mono- and biradicals produce abundant thiomethyl abstraction products and small amounts of DMDS radical cation, as expected. Surprisingly, all triradicals produce very abundant DMDS radical cations. A single-step mechanism involving electron transfer from DMDS to the triradicals is highly unlikely because the (experimental) adiabatic ionization energy of DMDS is almost 3 eV greater than the (calculated) adiabatic electron affinities of the triradicals. The unexpected reactivity can be explained based on an unprecedented two-step mechanism wherein the protonated triradical first transfers a proton to DMDS, which is then followed by hydrogen atom abstraction from the protonated sulfur atom in DMDS by the radical site in the benzene ring of the deprotonated triradical to generate the conventional DMDS radical cation and a neutral biradical. Quantum chemical calculations as well as examination of deuterated and methylated triradicals provide support for this mechanism. The proton affinities of the neutral triradicals (and DMDS) influence the first step of the reaction while the vertical electron affinities and spin-spin coupling of the neutral triradicals influence the second step. The calculated total reaction exothermicities for the triradicals studied range from 27.6 up to 29.9 kcal mol-1.

(-)ESI/CAD MSn Procedure for Sequencing Lignin Oligomers Based on a Study of Synthetic Model Compounds with ß-O-4 and 5-5 Linkages.

Seven synthesized G-lignin oligomer model compounds (ranging in size from dimers to an octamer) with 5-5 and/or ß-O-4 linkages, and three synthesized S-lignin model compounds (a dimer, trimer, and tetramer) with ß-O-4 linkages, were evaporated and deprotonated using negative-ion mode ESI in a linear quadrupole ion trap/Fourier transform ion cyclotron resonance mass spectrometer. The collision-activated dissociation (CAD) fragmentation patterns (obtained in MS2 and MS3 experiments, respectively) for the negative ions were studied to develop a procedure for sequencing unknown lignin oligomers. On the basis of the observed fragmentation patterns, the measured elemental compositions of the most abundant fragment ions, and quantum chemical calculations, the most important reaction pathways and likely mechanisms were delineated. Many of these reactions occur via charge-remote fragmentation mechanisms. Deprotonated compounds with only ß-O-4 linkages, or both 5-5 and ß-O-4 linkages, showed major 1,2-eliminations of neutral compounds containing one, two, or three aromatic rings. The most likely mechanisms for these reactions are charge-remote Maccoll and retro-ene eliminations resulting in the cleavage of a ß-O-4 linkage. Facile losses of H2O and CH2O were also observed for all deprotonated model compounds, which involve a previously published charge-driven mechanism. Characteristic "ion groups" and "key ions" were identified that, when combined with their CAD products (MS3 experiments), can be used to sequence unknown oligomers.

Laser-Induced Acoustic Desorption/Electron Ionization of Amino Acids and Small Peptides.

Laser-induced acoustic desorption (LIAD) allows for desorption of neutral nonvolatile compounds independent of their volatility or thermal stability. Many different ionization methods have been coupled with LIAD. Hence, this setup provides a better control over the types of ions formed than other mass spectrometry evaporation/ionization methods commonly used to characterize biomolecules, such as ESI or MALDI. In this study, the utility of LIAD coupled with electron ionization (EI) was tested for the analysis of common amino acids with no derivatization. The results compared favorably with previously reported EI mass spectra obtained using thermal desorption/EI. Further, LIAD/EI mass spectra collected for hydrochloride salts of two amino acids were found to be similar to those measured for the neutral amino acids with the exception of the appearance of an HCl+● ion. However, the hydrochloride salt of arginine showed a distinctly different LIAD/EI mass spectrum than the previously published literature EI mass spectrum, likely due to its highly basic side chain that makes a specific zwitterionic form particularly favorable. Finally, EI mass spectra were measured for seven small peptides, including di-, tri-, and tetrapeptides. These mass spectra show a variety of ion types. However, an type ions are prevalent. Also, electron-induced dissociation (EID) of protonated peptides has been reported to form primarily an type ions. In addition, the loss of small neutral molecules and side-chain cleavages were observed that are reminiscent of other high-energy fragmentation methods, such as EID. Finally, the isomeric dipeptides LG and IG were found to produce drastically different EI mass spectra, thus allowing differentiation of the leucine and isoleucine amino acids in these dipeptides. Graphical Abstract ᅟ.

Identification of the Phenol Functionality in Deprotonated Monomeric and Dimeric Lignin Degradation Products via Tandem Mass Spectrometry Based on Ion-Molecule Reactions with Diethylmethoxyborane.

Conversion of lignin into smaller molecules provides a promising alternate and sustainable source for the valuable chemicals currently derived from crude oil. Better understanding of the chemical composition of the resulting product mixtures is essential for the optimization of such conversion processes. However, these mixtures are complex and contain isomeric molecules with a wide variety of functionalities, which makes their characterization challenging. Tandem mass spectrometry based on ion-molecule reactions has proven to be a powerful tool in functional group identification and isomer differentiation for previously unknown compounds. This study demonstrates that the identification of the phenol functionality, the most commonly observed functionality in lignin degradation products, can be achieved via ion-molecule reactions between diethylmethoxyborane (DEMB) and the deprotonated analyte in the absence of strongly electron-withdrawing substituents in the ortho- and para-positions. Either a stable DEMB adduct or an adduct that has lost a methanol molecule (DEMB adduct-MeOH) is formed for these ions. Deprotonated phenols with an adjacent phenol or hydroxymethyl functionality or a conjugated carboxylic acid functionality can be identified based on the formation of DEMB adduct-MeOH. Deprotonated compounds not containing the phenol functionality and phenols containing an electron-withdrawing ortho- or para-substituent were found to be unreactive toward diethylmethoxyborane. Hence, certain deprotonated isomeric compounds with phenol and carboxylic acid, aldehyde, carboxylic acid ester, or nitro functionalities can be differentiated via these reactions. The above mass spectrometry method was successfully coupled with high-performance liquid chromatography for the analysis of a complex biomass degradation mixture. Graphical Abstract ᅟ.

Characterization of aromatic organosulfur model compounds relevant to fossil fuels by using atmospheric pressure chemical ionization with CS2 and high-resolution tandem mass spectrometry.

RATIONALE: The chemistry of desulfurization involved in processing crude oil is greatly dependent on the forms of sulfur in the oil. Sulfur exists in different chemical bonding environments in fossil fuels, including those in thiophenes and benzothiophenes, thiols, sulfides, and disulfides. In this study, the fragmentation behavior of the molecular ions of 17 aromatic organosulfur compounds with various functionalities was systematically investigated by using high-resolution tandem mass spectrometry. METHODS: Multiple-stage tandem mass spectrometric experiments were carried out using a linear quadrupole ion trap (LQIT) equipped with an atmospheric pressure chemical ionization (APCI) source. (+)APCI/CS2 was used to generate stable dominant molecular ions for all the compounds studied except for three sulfides that also showed abundant fragment ions. The LQIT coupled with an orbitrap mass spectrometer was used for elemental composition analysis, which facilitated the identification of the neutral molecules lost during fragmentation. RESULTS: The characteristic fragment ions generated in MS(2) and MS(3) experiments provide clues for the chemical bonding environment of sulfur atoms in the examined compounds. Upon collision-induced dissociation (CID), the molecular ions can lose the sulfur atom in a variety of ways, including as S (32 Da), HS(•) (33 Da), H2 S (34 Da), CS (44 Da), (•) CHS (45 Da) and CH2 S (46 Da). These neutral fragments are not only indicative of the presence of sulfur, but also of the type of sulfur present in the compound. Generally, losses of HS(•) and H2 S were found to be associated with compounds containing saturated sulfur functionalities, while losses of S, CS and (•) CHS were more common for heteroaromatic sulfur compounds. CONCLUSIONS: High-resolution tandem mass spectrometry with APCI/CS2 ionization is a viable approach to determining the types of organosulfur compounds. It can potentially be applied to analysis of complex mixtures, which is beneficial to improving the desulfurization process of fossil fuels. Copyright © 2016 John Wiley & Sons, Ltd.

Identification of N-Oxide and Sulfoxide Functionalities in Protonated Drug Metabolites by Using Ion-Molecule Reactions Followed by Collisionally Activated Dissociation in a Linear Quadrupole Ion Trap Mass Spectrometer.

The in vivo oxidation of sulfur and nitrogen atoms in many drugs into sulfoxide and N-oxide functionalities is a common biotransformation process. Unfortunately, the unambiguous identification of these metabolites can be challenging. In the present study, ion-molecule reactions of tris(dimethylamino)borane followed by collisionally activated dissociation (CAD) in an ion trap mass spectrometer are demonstrated to allow the identification of N-oxide and sulfoxide functionalities in protonated polyfunctional drug metabolites. Only ions with N-oxide or sulfoxide functionality formed diagnostic adducts that had lost dimethyl amine (DMA). This was demonstrated even for an analyte that contains a substantially more basic functionality than the functional group of interest. CAD of the diagnostic product ions (M) resulted mainly in type A (M - DMA) and B fragment ions (M - HO-B(N(CH3)2)2) for N-oxides, but sulfoxides also formed diagnostic C ions (M - O═BN(CH3)2), thus allowing differentiation of the functionalities. Some protonated analytes yielded abundant TDMAB adducts that had lost two DMA molecules instead of just one. This provides information on the environment of the N-oxide and sulfoxide functionalities. Quantum chemical calculations were performed to explore the mechanisms of the above-mentioned reactions. The method can be implemented on HPLC for real drug analysis.

Mass spectrometric studies of fast pyrolysis of cellulose.

A fast pyrolysis probe/linear quadrupole ion trap mass spectrometer combination was used to study the primary fast pyrolysis products (those that first leave the hot pyrolysis surface) of cellulose, cellobiose, cellotriose, cellotetraose, cellopentaose, and cellohexaose, as well as of cellobiosan, cellotriosan, and cellopentosan, at 600°C. Similar products with different branching ratios were found for the oligosaccharides and cellulose, as reported previously. However, identical products (with the exception of two) with similar branching ratios were measured for cellotriosan (and cellopentosan) and cellulose. This result demonstrates that cellotriosan is an excellent small-molecule surrogate for studies of the fast pyrolysis of cellulose and also that most fast pyrolysis products of cellulose do not originate from the reducing end. Based on several observations, the fast pyrolysis of cellulose is suggested to initiate predominantly via two competing processes: the formation of anhydro-oligosaccharides, such as cellobiosan, cellotriosan, and cellopentosan (major route), and the elimination of glycolaldehyde (or isomeric) units from the reducing end of oligosaccharides formed from cellulose during fast pyrolysis.

Multiported pulsed valve interface for a linear quadrupole ion trap mass spectrometer to enable rapid screening of multiple functional-group selective ion-molecule reactions.

Ion-molecule reactions provide a powerful tool for structural elucidation of ionized pharmaceutical analytes in tandem mass spectrometry. However, all previous interfaces for the introduction of reagents for ion-molecule reactions have utilized a single reagent approach. In this study, a multiported pulsed valve system was designed and characterized for rapid introduction of three neutral reagents into a linear quadrupole ion trap. Additionally, automatic triggering was used to allow for the introduction of the reagents on a chromatographic time scale. This system enables automatic, high throughput screening of complex mixtures by using at least three different ion-molecule reactions. Further, rapid testing of new neutral reagents is also possible.

Identification of the sulfone functionality in protonated analytes via ion/molecule reactions in a linear quadrupole ion trap mass spectrometer.

A tandem mass spectrometric method is presented for the rapid identification of drug metabolites that contain the sulfone functional group. This method is based on a gas-phase ion/molecule reaction of protonated sulfone analytes with trimethyl borate (TMB) that yields a diagnostic product ion, adduct-Me2O, at high reaction efficiency. A variety of compounds with different functional groups, such as sulfoxides, hydroxylamines, N-oxides, anilines, phenol, an aliphatic amine, and an aliphatic alcohol, were examined to probe the selectivity of this reaction. Except for protonated sulfones, most of the protonated compounds react very slowly or not at all with TMB. Most importantly, none of them give the adduct-Me2O product. A mechanism that explains the observed selectivity is proposed for the diagnostic reaction and is supported by quantum chemical calculations. The reaction was tested with the anti-inflammatory drug sulindac and its metabolite, sulindac sulfone, which were readily distinguished. The presence of other functionalities in addition to sulfone was found not to influence the diagnostic reactivity.