Quadrupole Linear Ion Trap Mass Research Articles

B-166 Differentiation of Aziridine Functionality from Related Functional Groups in Protonated Analytes by Using Selective Ion-Molecule Reactions

Abstract Background The development of better methods for the rapid and reliable identification of unknown compounds in mixtures is urgently needed in drug development. Aziridines, in particular, are used for drug synthesis but are potentially mutagenic and carcinogenic compounds. However, the detection and identification of these compounds pose challenges for many analytical methods, such as Nuclear Magnetic Resonance (NMR) spectroscopy, Fourier-transform infrared spectroscopy (FT-IR), and X-ray crystallography. These techniques are time-consuming and often require large quantities of highly pure compounds. Methods Therefore, this study demonstrates that selective gas-phase ion-molecule reactions of protonated analytes with neutral tris(dimethylamino)borane (TDMAB), followed by diagnostic collision-activated dissociation (CAD), can be used to differentiate aziridines from structurally related compounds in a modified linear quadrupole ion trap (LQIT) mass spectrometer. The experiments were performed using an atmospheric pressure chemical ionization (APCI) source in positive ion detection mode. TDMAB was purchased from Sigma-Aldrich and used without purification. It was introduced into the ion trap (2 mtorr of helium buffer gas) via an external reagent mixing manifold through a syringe pump at a flow rate of 10 μL h-1 and diluted with helium before entering the ion trap through a variable leak valve. All analytes were prepared at a concentration of 1 mM in methanol. The protonated analytes were transferred into the ion trap, isolated, and allowed to react with TDMAB (MS2 experiments). Results Product ions formed upon reactions of the analyte ions with TDMAB were subjected to a collision-activated dissociation (CAD) experiment (MS3 experiments). CAD on product ions produced diagnostic fragment ions with 25- and 43 m/z-units lower m/z values than the adduct - DMA product ions. Quantum chemical calculations were employed using the Gaussian 16 program calculated at the M06-2X/6-311++G(d,p) level of theory to provide theoretical foundation and to explore the underlying mechanisms of the observed gas-phase reactions. None of the tested structurally related analytes produced a similar fragmentation pattern Conclusions The innovative approach presented in this study offers a promising avenue for enhancing efficiency and accuracy in drug development. The successful differentiation of aziridine is an alternative method in overcoming the limitation of traditional techniques. The findings from the research contributes to the advancement of analytical techniques, addressing critical issues in identifying impurities in drug products. The versatility of the methods extends beyond drug developments, making it applicable in various other fields such as in molecular diagnostics, metabolism and laboratory medicines.

Characterization and comparison of metabolites in colostrum from yaks, buffaloes, and cows based on UPLC-QTRAP-MS metabolomics

Colostrum from yaks and buffaloes possesses substantial nutritional value, yet the complete array of metabolites within remains insufficiently elucidated. This study scrutinizes the metabolite profiles of yak, buffalo, and cow colostrum utilizing targeted metabolomics paired with ultra-performance liquid chromatography-tandem triple quadrupole linear ion trap mass spectrometry (UPLC-QTRAP-MS). The analysis detected 362 metabolites across all samples. Furthermore, 63, 77, and 46 differential metabolites were selected between yak and buffalo colostrum, yak and cow colostrum, and buffalo and cow colostrum, respectively. Yak colostrum notably contained higher concentrations of inositol, glycine, and carnitine, whereas buffalo colostrum was distinguished by a substantial presence of primary bile acids, which facilitate fat digestion. These findings offer profound insights into yak and buffalo colostrum, providing critical data to propel advancements in the dairy industry.

LC-MS based strategy for chemical profiling and quantification of dispensing granules of Ginkgo biloba seeds

Ginkgo biloba seeds have been used as a traditional Chinese medicine for hundreds of years to treat diseases such as cervicitis, cough, asthma and other lung diseases. As a novel form, the dispensing granules (GSDG) of Ginkgo biloba seeds have been widely employed in clinic. However, its chemical profiling is not yet clear, which has restricted in-depth research in many fields.In this study, a high performance liquid chromatography coupled with quardrupole time-of-flight mass spectrometry method was used for the component characteration with the help of accurate molecular weights, fragmentation pathways, reported data, literatures and even some reference standards. Furthermore, in multiple-reaction monitoring mode, a high performance liquid chromatography coupled with quadrupole linear ion trap mass spectrometry method was developed and applied for simultaneous determination of the bioactive phytochemicals.As a result, a total of 56 components in GSDG were identified including 12 amino acids, 9 organic acids, 6 nucleosides and nucleobases, 6 flavonoids, 5 vitamins, 5 terpenoid lactones, 4 carbohydrates and 9 other compounds As for quantitative analysis, glutamic acid, asparatic acid, histidine, ginkgolide A, ginkgolide B, ginkgolide C, ginkgolide J, eucomic acid, N-(N-glucopyranosyl)-indoleacetylaspartate and N-(N-glucopyranosyl)-indoleacetylglutamate were selected as the analytes for quanlity marker of GSDG. After necessary validation tests, the developed quantitative method was successfully put into use for 10 batches of GSDG. In all batches, N-(N-glucopyranosyl)-indoleacetylaspartate was the richest phytochemical with the amount of 17.3–25.7 mg/g while ginkgolide J (0.0197–0.0335 mg/g) was determined to be the poorest.The study is supposed to exhibit a comprehensive chemical profiling and to provide some strong basis for preparation technology, quality control and even for action mechanism of GSDG, this novel form of Chinese medicine.

Study on the Mass Spectrometry Cleavage Pattern of Quinolone Antibiotics.

Quinolone antibiotics are extensively used clinically for human treatment and in agriculture. However, improper and excessive use can lead to the persistence of quinolone residues in animal tissues, potentially accumulating in the human body and posing health risks. Investigating the correlation between mass spectrometry cleavage patterns and molecular structural features enhances the analytical framework for detecting trace or unknown impurities in quinolones. To collect data, we employed triple quadrupole linear ion trap mass spectrometry in electrospray positive ion mode. Primary mass spectrometry scanning was utilized to confirm parent ions, while secondary mass spectrometry scanning enabled the observation of fragment ions. The cleavage characteristics and pathways of the compounds were inferred from accurate mass-to-charge ratios obtained from both primary and secondary mass spectrometry. Under soft ionization conditions, the compounds generally exhibited characteristic fragment ions of [M+H-H2O]+, [M+H-CO]+, and [M+H-H2O-CO]+. Additionally, subtle variations were observed in each compound due to differences in modifying groups. For instance, upon deacidification, the piperazine ring structure underwent breakage and rearrangement, yielding fragment ion peaks devoid of neutral molecules such as C2H5N, C3H7N, or C4H8N. Notably, compounds featuring a cyclopropyl substituent group at the N-1 position typically exhibited characteristic fragments resulting from the loss of the cyclopropyl radical (⋅C3H5). Moreover, substituents at the N-1 and C-8 positions, when linked to form a six-membered carbocyclic ring, were prone to cleavage, releasing the neutral C3H6 molecule. Quinolone antibiotics share structural similarities in their parent nuclei, leading to partially similar cleavage pathways. Nevertheless, distinct cleavage patterns emerge due to variations in functional groups. According to the difference of mass spectrometry cleavage patterns, it can provide an identification basis for the measured detection of antibiotics.

Pseudotargeted metabolomics method and its application in erastin-stimulated gastric adenocarcinoma cells based on liquid chromatography with tandem mass spectrometry

Erastin, a classical ferroptosis inducer, exerts cytotoxicity in several types of cancer cells including gastric cancer cells. However, the mechanism of erastin in regulating metabolic pathways in gastric cancer remains largely unclear. To investigate the gastric cellular response to erastin therapy, a pseudotargeted metabolomics method was achieved on ultra-high performance liquid chromatography-hybrid triple quadrupole linear ion trap mass spectrometry (UHPLC-QTRAP MS), which was used to investigate metabolic changes between erastin-treated MGC-803 cells and the controls at different time points. We found that erastin induced tremendous impact on the metabolome of gastric cells by affecting key metabolic processes, such as cysteine and methionine metabolism, tryptophan metabolism, purine metabolism, glutathione biosynthesis, glycolysis and TCA cycle. Interestingly, S-adenosylmethionine, methionine, serine and cysteine were obviously increasing treads after erastin treatment, while S-adenosylhomocysteine and glutathione were always down-regulated up to 24 h. The results indicated that DNA methylation was activated and glutathione biosynthesis was blocked in erastin-treated MGC-803 gastric cells, highlighting the importance of erastin as a promising drug candidate for in vivo treatment of gastric tumor.

Liquid chromatography-mass spectrometry-based strategy for systematic profiling of chemical components and associated quantitative analysis of quality markers in Qi-Wei-Tong-Bi oral liquid.

Qi-Wei-Tong-Bi oral liquid (QWTB), a famous Chinese medicine preparation composed of seven crude drugs has a good therapeutic effect on rheumatoid arthritis and is widely used in China.However, its chemical composition and quality control have not been comprehensively and systematically investigated. In this study, high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry was employed for its chemical profiling. As a result, 100 components were chemically characterized. Additionally, high-performance liquid chromatography coupled with a quadrupole linear ion trap mass spectrometry method was developed to simultaneously quantify nine bioactive components (hyperoside, ononin, quercetin, sinomenine, magnoflorine, gallic acid, protocatechuic acid, monotropein, and cyclo-(Pro-Tyr)) in multiple-reaction monitoring mode. After successful validation in terms of linearity, precision, repeatability, and recovery, the assay method was applied for the determination of 10 batches of QWTB. The results showed that QWTB was enriched in sinomenine and magnoflorine with the highest amount up to hundreds or even thousands of µg/mL, while quercetin, ononin, cyclo-(Pro-Tyr), and hyperoside were much lower with the lowest content below 10µg/mL. This study work would help to reveal the chemical profiling and provide a valuable and reliable approach for quality evaluation and even pharmacodynamic material basis studies of QWTB.

LC-MS/MS-MS3 for the Determination and Quantification of Δ9-Tetrahydrocannabinol and Metabolites in Blood Samples.

Due to the high prevalence of cannabinoids in forensic toxicology analysis, it is crucial to have an efficient method that allows the use of a small sample amount and that requires a minimal sample preparation, for the determination and quantification of low concentrations. A simple, highly selective and high throughput liquid chromatography tandem mass spectrometry methodology (LC-MS/MS-MS3) was developed for the determination and quantification of ∆9-tetrahydrocannabinol (THC), 11-hydroxy-∆9- tetrahydrocannabinol (THC-OH) and 11-nor-9-carboxy-∆9-tetrahydrocannabinol (THC-COOH), in blood samples. Chromatographic analysis of THC, THC-OH, and THC-COOH and their deuterated internal standards was preceded by protein precipitation (PPT) of 0.1 mL of blood samples with acetonitrile. Chromatographic separation was achieved by use of an Acquity UPLC® HHS T3 (100 mm x 2.1mm i.d., 1.8 μm) reversed-phase column, using a gradient elution of 2 mM aqueous ammonium formate, 0.1% formic acid, and methanol at a flow rate of 0.4 mL/min, with a run time of 10 minutes. For the MS/MS-MS3 analysis, a SCIEX QTRAP® 6500+ triple quadrupole linear ion trap mass spectrometer was used via electrospray ionization (ESI), operated in multiple reaction monitoring (MRM) and linear ion trap mode (MS3). The method was validated in accordance with internationally accepted criteria and guidelines, and proved to be selective and linear between 0.5-100 ng/mL (r2>0.995). The lower limits of quantification (LLOQ) corresponded to the lowest concentrations used for the calibration curves. The coefficients of variation obtained for accuracy and precision were <15%. The mean recoveries were between 88.0-117.2%, for the studied concentration levels (1 ng/mL, 5 ng/mL and 50 ng/mL). No significant interfering compounds, matrix effects or carryover were observed. The validated method provides a sensitive, efficient and robust procedure for the quantification of cannabinoids in blood, using LC-MS/MS-MS3 and a sample volume of 0.1 mL. This work is also a proof of concept for using LC-MS3 technique to determine drugs in biological samples.

Differentiation of the Aziridine Functionality from Related Functional Groups in Protonated Analytes by Using Selective Ion-Molecule Reactions Followed by Collision-Activated Dissociation in a Linear Quadrupole Ion Trap Mass Spectrometer.

Aziridines are commonly used as reagents for the synthesis of drug substances although they are potentially mutagenic and genotoxic. Therefore, their unambiguous detection is critically important. Unfortunately, tandem mass spectrometry (MS2) based on collision-activated dissociation (CAD), a powerful method used for the identification of many unknown compounds in complex mixtures, does not provide diagnostic fragmentation patterns for ionized aziridines. Therefore, a different mass spectrometry approach based on MS3 experiments is presented here for the identification of the aziridine functionalities. This approach is based on selective gas-phase ion-molecule reactions of protonated analytes with tris(dimethylamino)borane (TDMAB) followed by diagnostic CAD reactions in a modified linear quadrupole ion trap (LQIT) mass spectrometer. TDMAB reacts with protonated aziridines by forming adduct ions that have lost a dimethylamine (DMA) molecule ([M + H + TDMAB - HN(CH3)2]+). CAD on these product ions generated diagnostic fragment ions with m/z-values 25- and 43-units lower than those of the ion-molecule reaction product ions. None of the ion-molecule reaction product ions formed from other, structurally related, protonated analytes produced related fragment ions. Quantum chemical calculations were employed to explore the mechanisms of the observed reactions.

Improved analysis of folpet and captan in foods using liquid chromatography-triple quadrupole linear ion trap mass spectrometry: applying mass filtering, collision, and trapping conditions

Accurate and highly sensitive analysis of folpet and captan was accomplished using liquid chromatography-triple quadrupole linear ion trap mass spectrometry (LC-QqQIT) with selective ion mode; mass filtering, collision, and trapping condition. Dimensional mass spectrometry (MS3) parameters were optimized for the residue detection of folpet and captan in six food commodities (apples, tomatoes, sweet pepper, wheat flour, sesame seeds, and fennel seeds). The sample preparation method was based on the known QuEChERS protocol, except a mixture of acetonitrile/acetone was used for the sample extraction from the sesame seeds. The robustness and reliability of the developed MS3 method were demonstrated by performing a full validation, according to SANTE/11312/2021, at 0.01-0.25mg/kg. Recovery ranged from 83 to 118% with a relative standard deviation below 19% in all the tested commodities, and limits of quantifications (LOQs) were 0.01mg/kg in apples and tomatoes; 0.03mg/kg in sweet pepper; and 0.05mg/kg in wheat flour, sesame seeds, and fennel seeds. Monitoring results showed that about 90% of apples contained captan residue, and in sweet pepper, concentrations of captan and folpet as high as 1.57 and 0.97mg/kg were found, respectively. The novel developed MS3 method enabled more reliable identification of these commonly problematic fungicides at lower LOQs than previously reported methods.

Comparative pharmacokinetic study of nine bioactive components in osteoarthritis rat plasma using ultra-performance liquid chromatography-tandem mass spectrometry after single and combined oral administration of Epimedii Folium and Chuanxiong Rhizoma extracts.

The herb pair Epimedii Folium-Chuanxiong Rhizoma (EF-CR), derived from the classical traditional Chinese medicine 'Xian Ling Pi San', has a distinctive compatibility therapeutic profile and is clinically safe and effective. This study aimed to investigate and compare the pharmacokinetic characteristics of nine analytes in osteoarthritis (OA) rat plasma after the oral administration of EF, CR or a combination of these two herbs. We developed an ultra-performance liquid chromatography method coupled with quadrupole linear ion-trap mass spectrometry to simultaneously quantify and assess the pharmacokinetics of icariin, epimedin A, epimedin B, epimedin C, icariside I, icariside II, ferulic acid, ligustilide and senkyunolide A of the EF-CR pair in the plasma of osteoarthritic rats. The pharmacokinetic parameters showed that the absorption of multiple components was significantly enhanced and residence time was prolonged in the EF-CR group (P < 0.05) compared to the single-herb group. These parameters revealed that the combination of EF and CR exhibited synergistic effects of the nine bioactive components, suggesting the potential application of the EF-CR combination for the treatment of OA.

A Diagnostic Nitrosamine Detection Approach for Pharmaceuticals by Using Tandem Mass Spectrometry Based on Diagnostic Gas-Phase Ion-Molecule Reactions.

N-Nitrosamines are strictly regulated in pharmaceutical products due to their carcinogenic nature. Therefore, the ability to rapidly and reliably identify the N-nitroso functionality is urgently needed. Unfortunately, not all ionized N-nitroso compounds produce diagnostic fragment ions and hence tandem mass spectrometry based on collision-activated dissociation (CAD) cannot be used to consistently identify the N-nitroso functionality. Therefore, a more reliable method was developed based on diagnostic functional-group selective ion-molecule reactions in a linear quadrupole ion trap mass spectrometer. 2-Methoxypropene (MOP) was identified as a reagent that reacts with protonated N-nitrosamines in a diagnostic manner by forming an adduct followed by the elimination of 2-propenol (CH3C(OH)═CH2]). From 18 protonated N-nitrosamine model compounds studied, 15 formed the diagnostic product ion. The lack of the diagnostic reaction for three of the N-nitrosamine model compounds was rationalized based on the presence of a pyridine ring that gets preferentially protonated instead of the N-nitroso functionality. These N-nitrosamines can be identified by subjecting a stable adduct formed upon ion-molecule reactions with MOP to CAD. Further, the ability to use ion-molecule reactions followed by CAD to differentiate protonated O-nitroso compounds with a pyridine ring from analogous N-nitrosamines was demonstrated This methodology is considered to be robust for the identification of the N-nitroso functionality in unknown analytes. Lastly, HPLC/MS2 experiments were performed to determine the detection limit for five FDA regulated N-nitrosamines.

Proton Affinities of Alkanes.

Chemical characterization of complex mixtures of large alkanes is critically important for many fields, including petroleomics and the development of renewable transportation fuels. Tandem mass spectrometry is the only analytical method that can be used to characterize such mixtures at the molecular level. Many ionization methods used in mass spectrometry involve proton transfer to the analyte. Unfortunately, very few proton affinity (PA) values are available for alkanes. Indeed, previous research has shown that most protonated alkanes (MH+) are not stable but fragment spontaneously via the elimination of a hydrogen molecule to form [M - H]+ ions. Here, the PAs of several n-alkanes and alkylcyclohexanes containing 5-8 carbon atoms, n-pentane, n-hexane, n-heptane, n-octane, cyclohexane, methylcyclohexane, and ethylcyclohexane, were determined via bracketing experiments by using a linear quadrupole ion trap mass spectrometer. Monitoring the formation of the [M - H]+ ions in reactions between the alkanes and protonated reference bases with known PAs revealed that the PAs of all the alkanes fell into the range 721 ± 20 kJ mol-1. In order to obtain a more accurate estimate of the relative PAs of different alkanes, two alkanes were introduced simultaneously into the ion trap and allowed to react with the same protonated reference base. Based on these experiments, the longer the alkyl chain in an n-alkane or alkylcyclohexane the greater the PA. Further, when considering alkanes with the same number of carbon atoms, the PAs of those with a cyclohexane ring were found to be greater than those with no such ring.

A Portable Reagent Inlet System Designed to Diminish the Impact of Air and Water to Ion-Molecule Reactions Studied in a Linear Quadrupole Ion Trap.

A portable reagent inlet system for a linear quadrupole ion trap (LQIT) mass spectrometer was designed to diminish the impact of air and water on gas-phase ion-molecule reactions. Compared to the traditional reagent mixing manifolds that has been extensively used for decades, the portable system is much simpler and has fewer junctions and a smaller inner space. These changes reduce the amount of air and water introduced into the mass spectrometer with the reagent. Furthermore, unlike the traditional manifolds, the portable system can be easily attached to or detached from the LQIT mass spectrometer. Finally, the price of the portable system is only 1/10 of that of a traditional manifold as estimated in 2022. Therefore, the portable system has several advantages over the traditional reagent mixing manifolds.

Tandem Mass Spectrometric Characterization of the Molecular Radical Cations of Asphaltenes

Asphaltenes, the heaviest fraction of crude oil, are problematic for the oil industry for several reasons. For example, they have a tendency to precipitate out of oil pipelines, thus causing clogs. Further, they can foul catalysts used in refining of crude oil. However, the lack of knowledge on the exact molecular structures of asphaltenes has long prevented addressing the above problems and also hindered finding financially beneficial uses for asphaltenes. This paper focuses on recent advances on the structural characterization of the molecular radical cations of asphaltenes based on collision-activated dissociation (CAD) experiments performed using linear quadrupole ion trap mass spectrometers coupled to an Orbitrap detector. Stable molecular radical cations are readily generated from asphaltenes upon atmospheric pressure chemical ionization (APCI) when using CS2 as the solvent because this solvent cannot transfer a proton to analytes. MS2–MS5 experiments based on ion-source CAD (ISCAD), ion-trap CAD (ITCAD), and/or medium-energy CAD (MCAD) were explored for the structural characterization of these molecular radical cations by using model compounds. Energy-resolved MCAD experiments were identified as the most useful experiments because they provide substantially more structural information for the radical cations than the other CAD experiments mentioned above. In some MSn experiments, MSn–1 was first performed in the ion trap, followed by isolation of a desired ion in the ion trap and transfer into the MCAD collision cell, wherein it was subjected to energy-resolved MCAD followed by high-resolution detection of the fragment ions. The various CAD experiments were used to obtain information on compounds in asphaltene samples with different origins, such as the aliphatic carbon content, molecular weight distributions (MWDs), average molecular weight, number of aromatic carbon atoms, number of aromatic rings in the core, and relative abundances of single-core and multicore compounds.

Changes in Phospholipid Composition of the Human Cerebellum and Motor Cortex during Normal Ageing.

(1) Background: Changes in phospholipid (phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine, i.e., PC, PE and PS) composition with age in the mitochondrial and microsomal membranes of the human cerebellum and motor cortex were examined and compared to previous analyses of the prefrontal cortex, hippocampus and entorhinal cortex. (2) Methods: Nano-electrospray ionization on a hybrid triple quadrupole–linear ion trap mass spectrometer was used to analyse the brain regions of subjects aged 18–104 years. (3) Results: With age, the cerebellum showed many changes in the major phospholipids (>10% of the phospholipid class). In both membrane types, these included increases in PE 18:0_22:6 and PS 18:0_22:6, decreases in PE 18:0_20:4 and PS 18:0_18:1 and an increase in PC 16:0_16:0 (microsomal membrane only). In addition, twenty-one minor phospholipids also changed. In the motor cortex, only ten minor phospholipids changed with age. With age, the acyl composition of the membranes in the cerebellum increased in docosahexaenoic acid (22:6) and decreased in the arachidonic (20:4) and adrenic (22:4) acids. A comparison of phospholipid changes in the cerebellum, motor cortex and other brain areas is provided. (4) Conclusions: The cerebellum is exceptional in the large number of major phospholipids that undergo changes (with consequential changes in acyl composition) with age, whereas the motor cortex is highly resistant to change.

Systematic Qualitative and Quantitative Analyses of Wenxin Granule via Ultra-High Performance Liquid Chromatography Coupled with Ion Mobility Quadrupole Time-of-Flight Mass Spectrometry and Triple Quadrupole-Linear Ion Trap Mass Spectrometry.

Wenxin granule (WXG) is a popular traditional Chinese medicine (TCM) preparation for the treatment of arrhythmia disease. Potent analytical technologies are needed to elucidate its chemical composition and assess the quality differences among multibatch samples. In this work, both a multicomponent characterization and quantitative assay of WXG were conducted using two liquid chromatography–mass spectrometry (LC-MS) approaches. An ultra-high performance liquid chromatography–ion mobility quadrupole time-of-flight mass spectrometry (UHPLC/IM-QTOF-MS) approach combined with intelligent peak annotation workflows was developed to characterize the multicomponents of WXG. A hybrid scan approach enabling alternative data-independent and data-dependent acquisitions was established. We characterized 205 components, including 92 ginsenosides, 53 steroidal saponins, 14 alkaloids, and 46 others. Moreover, an optimized scheduled multiple reaction monitoring (sMRM) method was elaborated, targeting 24 compounds of WXG via ultra-high performance liquid chromatography–triple quadrupole linear ion trap mass spectrometry (UHPLC/QTrap-MS), which was validated based on its selectivity, precision, stability, repeatability, linearity, sensitivity, recovery, and matrix effect. By applying this method to 27 batches of WXG samples, the content variations of multiple markers from Notoginseng Radix et Rhizoma (21) and Codonopsis Radix (3) were depicted. Conclusively, we achieved the comprehensive multicomponent characterization and holistic quality assessment of WXG by targeting the non-volatile components.

Gas-Phase Reactivity of Phenylcarbyne Anions.

Gas-phase reactivities of the phenylcarbyne anion and its four derivatives were studied using a linear quadrupole ion trap mass spectrometer. The phenylcarbyne anions were calculated to have a triplet ground state (singlet-triplet splittings of 4-9 kcal mol-1), with the exception of the 4-cyanophenylcarbyne anion that has a singlet ground state (singlet-triplet splitting of -1.9 kcal mol-1). Only the phenylcarbyne anions with a triplet ground state react with acetone and dimethyl disulfide via radical mechanisms. On the other hand, only the phenylcarbyne anion with a singlet ground state abstracts H2O and H2C═C═O from acetic acid via electrophilic addition of the reagents to the anion. Finally, two hydroxy-substituted phenylcarbyne anions (with triplet ground states) partially tautomerize with the assistance of reagent molecules to the more stable distonic phenylcarbene anions. This occurs via abstraction of a proton from the reagent by the phenylcarbyne anion to generate a neutral (triplet) phenylcarbene and a reagent anion, which is followed by proton abstraction from the hydroxyl group of the neutral phenylcarbene by the reagent anion to generate the distonic phenylcarbene anion in an excited triplet state. Experiments performed on deuterated hydroxy-substituted phenylcarbyne anions verified the mechanism. The reactivities of the distonic phenylcarbene anions were found to be quite different from those of the phenylcarbyne anions. For example, they were found to abstract CH2 from acetonitrile, which is initiated by C-H insertion─typical singlet carbene reactivity.

Fluoropyrimidine Bioactivation and Metabolism by the Gut Microbiome

Despite decades of research into why populations of cancer patients differentially respond to the same treatment, variation in response to therapy regimens remains a significant challenge in the oncology field. However, our understanding of the causal mechanisms of interpatient variability remains lacking; up to 35% of patients require dose adjustments and 10% of patients are removed from the fluoropyrimidine capecitabine altogether. This research seeks to further our understanding of the microbiome’s role in fluoropyrimidine treatment by identifying gut bacterial strains responsible for capecitabine activation as well as biochemically characterizing drug inactivating enzymes from the human gut microbiome. Growth curve data show that knockouts of bacterial enzymes homologous to mammalian enzymes known to be involved in capecitabine metabolism increase bacterial resistance to capecitabine. Additionally, an unbiased transposon mutagenesis screen identifies enzymes known to increase resistance as well as enzymes both within and outside of nucleotide metabolism. These two pieces of data suggest that bacteria have the capacity to metabolize capecitabine, upending the paradigm that activation of this prodrug happens exclusively in the liver and tumor. Confirmation of these metabolites with high resolution triple quadrupole linear ion trap mass spectrometry is ongoing. Separately, previous studies indicate that gut bacteria are capable of degrading 5‐fluorouracil, however it is not clear how efficient these pathways are or to what extent they occur in the host. Enzyme kinetics of homologues of this enzyme within the microbiome show that degradative reaction for uracil is essentially irreversible. Additionally, this enzymatic characterization has identified the non‐enzymatic enantiomer of the degraded fluoropyrimidine as a potential inhibitor for this enzyme, which might allow for co‐dosing and inhibition of bacterial metabolism. Metagenomic sequencing of the degradative enzymes within patient samples is ongoing and will potentially reveal the main drivers of this activity in patients. These avenues of research will uncover new bacterial metabolism pathways of xenobiotics, compare these metabolic pathways between microbiota and host, and have the potential to lead to new advances in disrupting microbial metabolism of chemotherapeutics.

Mechanospray Ionization MS of Proteins Including in the Folded State and Polymers.

Mechanospray ionization (MoSI) is a technique that produces ions directly from solution-like electrospray ionization (ESI) but without the need of a high voltage. In MoSI, mechanical vibrations aerosolize solution phase analytes, whereby the resulting microdroplets can be directed into the inlet orifice of a mass spectrometer. In this work, MoSI is applied to biomolecules up to 80 kDa in mass in both denatured and native conditions as well as polymers up to 12 kDa in mass. The various MoSI devices used in these analyses were all comprised of a piezoelectric annulus attached to a central metallic disk containing an array of 4 to 7 μm diameter holes. The devices vibrated in the 100-170 kHz range to generate a beam of microdroplets that ultimately resulted in ion formation. A linear quadrupole ion trap (LIT) and orbitrap mass spectrometer were used in the analysis to investigate higher mass proteins at both native (folded) and denatured (unfolded) conditions. MoSI native mass spectra of proteins acquired on the orbitrap and LIT instrument demonstrated that proteins could remain intact and in a folded state. In the case of native MS of holomyoglobin, the intact folded protein remained mostly bound noncovalently to the heme group, and typically, the spectra showed reduced loss of the heme group by MoSI as compared to ESI. In both non-native and native protein analyses examples, broader often multimodal distributions to lower charge states were observed. When using the LIT instrument, a significant increase in the relative abundance of dimers was observed by MoSI as compared to ESI. The softness of the MoSI technique was evidenced by the lack of fragmentation, the formation of dimers as also noted by others ( J. Mass Spectrom. 2016, 424-429) and under native conditions, the retention of proteins in one or more presumed folded structures and for holomyoglobin the high retention of the heme group. When analyzing polyethylene glycol (PEG) and polypropylene glycol (PPG), MoSI also generated a broader distribution to lower charge states than ESI. By using the improved separation of peaks at lower charge states and all the charge states available, MoSI data should provide an improved ionization method to obtain more accurate mass and dispersity values for some polymers.

Chiral analysis of dextromethorphan and levomethorphan in human hair by liquid chromatography-tandem mass spectrometry.

Methorphan exists in two enantiomeric forms including dextromethorphan and levomethorphan. Dextromethorphan is an over-the-counter antitussive drug, whereas levomethorphan is strictly controlled as a narcotic drug. Chiral analysis of methorphan could, therefore, assist clinicians and forensic experts in differentiating between illicit and therapeutic use and in tracing the source of the drug. A method for enantiomeric separation and quantification of levomethorphan and dextromethorphan in human hair was developed and validated using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Hair was extracted in hydrochloric acid/methanol (1:20, v/v). The supernatant were separated using a Supelco Astec Chirobiotic™ V2 column (250 × 2.1mm, i.d., 5μm particle size) and analyzed on a triple quadrupole linear ion trap mass spectrometer in multiple reaction monitoring mode. The limits of detection for dextromethorphan and levomethorphan were 2 and 1pg/mg, respectively; the lower limit of quantification was 2pg/mg for both drugs. Good linearity (r > 0.995) was observed for both analytes over the linear range. Precision values were below 10% for both analytes; accuracy values ranged from 87.5 to 101%. The extraction recoveries were 78.3-98.4%, and matrix effects were 70.5-88.6%. This method was applied to human hair samples from 120 people suspected of methorphan use to further distinguish the drug chirality. Dextromethorphan was detected in all 120 samples at a concentration range of 2.7-19,100pg/mg, whereas levomethorphan was not detected in any sample. A sensitive quantitative method was established for the enantiomeric separation of dextromethorphan and levomethorphan in hair. This is the first study to achieve chiral analysis of methorphan in human hair.