Electrospray Ionization Quadrupole Time-of-flight Research Articles

Detecting M-Protein via Mass Spectrometry and Affinity Beads: Enrichment With Mixed Kappa-Lambda Beads Enables Prompt Application in Clinical Laboratories.

Detecting monoclonal protein (M-protein), a hallmark of plasma cell disorders, traditionally relies on methods such as protein electrophoresis, immune-electrophoresis, and immunofixation electrophoresis (IFE). Mass spectrometry (MS)-based methods, such as matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and electrospray ionization-quadrupole time-of-flight (ESI-qTOF) MS, have emerged as sensitive methods. We explored the M-protein-detection efficacies of different MS techniques. To isolate immunoglobulin and light chain proteins, six types of beads (IgG, IgA, IgM, kappa, lambda, and mixed kappa and lambda) were used to prepare samples along with CaptureSelect nanobody affinity beads (NBs). After purification, both MALDI-TOF MS and liquid chromatography coupled with Synapt G2 ESI-qTOF high-resolution MS analysis were performed. We purified 25 normal and 25 abnormal IFE samples using NBs and MALDI-TOF MS (NB-MALDI-TOF). Abnormal samples showed monoclonal peaks, whereas normal samples showed polyclonal peaks. The IgG and mixed kappa and lambda beads showed monoclonal peaks following the use of daratumumab (an IgG/kappa type of monoclonal antibody) with both MALDI-TOF and ESI-qTOF MS analysis. The limits of detection for MALDI-TOF MS and ESI-qTOF MS were established as 0.1 g/dL and 0.025 g/dL, respectively. NB-MALDI-TOF and IFE exhibited comparable sensitivity and specificity (92% and 92%, respectively). NBs for M-protein detection, particularly with mixed kappa-lambda beads, identified monoclonal peaks with both MALDI-TOF and ESI-qTOF analyses. Qualitative analysis using MALDI-TOF yielded results comparable with that of IFE. NB-MALDI-TOF might be used as an alternative method to replace conventional tests (such as IFE) to detect M-protein with high sensitivity.

Comprehensive polyoxypregnane glycosides report in Caralluma quadrangula using UPLC–ESI–Q–TOF and their antioxidant effects in human plasma

Caralluma quadrangula (Forssk.) N.E.Br. (Syns: = Stapelia quadrangula Forssk. = Monolluma quadrangula Forssk.) is an indigenous member of the genus Caralluma and it is a rather common species on rocky hillsides in the southwestern part of Saudi Arabia. Several members of this genus have found medicinal uses in the treatment of rheumatism, diabetes, leprosy and as antiseptics and disinfectants. All parts are edible but rather more bitter and can cause diarrhea.The present report was tentatively elucidated the structure of acylated and non-acylated polyoxypregnane glycosides from Caralluma quadrangula.The analyses were performed using an electrospray-ionization quadrupole time-of-flight (ESI-Q-TOF) mass spectrometer in both positive and negative ionization modes to explore fragmentation pathways. The antioxidant and prooxidant properties of the different mobility portions of human plasma were evaluated in vitro using thiobarbituric acid reactive substance assay (TBARS).The analyses showed sixty-five characteristic ion peaks which could be more efficient to assignment the aglycones and fragmentation sequences of sugar moieties. The used ionization modes provided consistent and/or complementary information for most of the pregnane glycosides, their fragmentation sequences, and their aglycones. A DFT Study was performed to elucidate the neutral loss of H2O molecules sequences from aglycones and the esterification linkage.This report could be useful to reduce material consuming and time in phytochemistry analysis of the different medicinal plants. The two portions significantly depleted TBARS were subjected to autoperoxidation assay in the presence of hydrogen peroxide.

Characterization of Potential Protein Biomarkers for Major Depressive Disorder Using Matrix-Assisted Laser Desorption Ionization/Time-of-Flight Mass Spectrometry

Matrix-assisted laser desorption ionization/time-of-flight (MALDI-TOF) mass spectrometry is a sensitive analytical tool for characterizing various biomolecules in biofluids. In this study, MALDI-TOF was used to characterize potential plasma biomarkers for distinguishing patients with major depressive disorder (MDD) from patients with schizophrenia and healthy controls. To avoid interference from albumin—the predominant protein in plasma—the plasma samples were pretreated using acid hydrolysis. The results obtained by MALDI-TOF were also validated by electrospray ionization-quadrupole time-of-flight (ESI-QTOF) mass spectrometry. The analytical results were further treated with principal component analysis (PCA), hierarchical clustering analysis (HCA), and receiver operating characteristic (ROC) curve analysis. The statistical analyses showed that MDD patients could be distinguished from schizophrenia patients and healthy controls by the lack of apolipoprotein C1 (Apo C1), which, in fact, was detected in healthy controls and schizophrenia patients. This protein is suggested to be a potential plasma biomarker for distinguishing MDD patients from healthy controls and schizophrenia patients. Since sample preparation for MALDI-TOF is very simple, high-throughput plasma apolipoprotein analysis for clinical purposes is feasible.

Comparison of in-source collision-induced dissociation and beam-type collision-induced dissociation of emerging synthetic drugs using a high-resolution quadrupole time-of-flight mass spectrometer.

In-source collision-induced dissociation (CID) is commonly used with single-stage high-resolution mass spectrometers to gather both a molecular formula and structural information through the collisional activation of analytes with residual background gas in the source region of the mass spectrometer. However, unlike tandem mass spectrometry, in-source CID does not involve an isolation step prior to collisional activation leading to a product ion spectrum composed of fragment ions from any analyte present during the activation event. This work provides the first comparison of in-source CID and beam-type CID spectra of emerging synthetic drugs on the same instrument to understand the fragmentation differences between the two techniques and to contribute to the scientific foundations of in-source CID. Electrospray ionization-quadrupole time-of-flight (ESI-Q-TOF) mass spectrometry was used to generate product ion spectra from in-source CID and beam-type CID for a series of well-characterized fentanyl analogs and synthetic cathinones. A comparison between the fragmentation patterns and relative ion abundances for each technique was performed over a range of fragmentor offset voltages for in-source CID and a range of collision energies for beam-type CID. The results indicate that large fragmentor potentials for in-source CID tend to favor higher energy fragmentation pathways that result in both kinetically favored pathways and consecutive neutral losses, both of which produce more abundant lower mass product ions relative to beam-type CID. Although conditions can be found in which in-source CID and beam-type CID provide similar overall spectra, the in-source CID spectra tend to contain elevated noise and additional chemical background peaks relative to beam-type CID.

Reversible Oxygen Sensing Based on Multi-Emission Fluorescence Quenching.

Oxygen is ubiquitous in nature and it plays a key role in several biological processes, such as cellular respiration and food deterioration, to name a few. Currently, reversible and non-destructive oxygen sensing is usually performed with sensors produced by photosensitization of phosphorescent organometallic complexes. In contrast, we propose a novel route of optical oxygen sensing by fluorescence-based quenching of oxygen. We hereby developed for the first time a set of multi-emissive purely organic emitters. These were produced through a one-pot hydrothermal synthesis using p-phenylenediamine (PPD) and urea as starting materials. The origin of the multi-emission has been ascribed to the diversity of chemical structures produced as a result of oxidative oligomerization of PPD. A Bandrowski’s base (BB, i.e., trimer of PPD) is reported as the main component at reaction times higher than 8 h. This indication was confirmed by electrospray-ionization quadrupole time-of-flight (ESI-QTOF) and liquid chromatography-mass spectrometry (LC-MS) analysis. Once the emitters are embedded within a high molecular weight poly (vinyl alcohol) matrix, the intensities of all three emission centers exhibit a non-linear quenching provoked by oxygen within the range of 0–8 kPa. The detection limit of the emission centers are 0.89 kPa, 0.67 kPa and 0.75 kPa, respectively. This oxygen-dependent change in fluorescence emission is reversible (up to three tested 0–21% O2 cycles) and reproducible with negligible cross-interference to humidity. The cost-effectiveness, metal-free formulation, cross-referencing between each single emission center and the relevant oxygen range are all appealing features, making these sensors promising for the detection of oxygen, e.g., in food packaged products.

Stability study and oxidative degradation kinetics of posaconazole

Posaconazole (POS), a triazole derivative used for the treatment of systemic fungal infections, was recently found to be a potential therapeutic alternative for patients with chronic Chagas disease. It is necessary to determine the stability of POS and identify its degradation products to achieve the expected pharmacological effects and assess the risks of drug toxicity associated with this novel therapeutic application. This study aimed to evaluate POS stability under varied conditions using forced degradation tests (acidic and basic hydrolysis, thermolysis, and oxidation). A high-performance liquid chromatography (HPLC) method was developed for the separation and identification of POS and its degradation products. POS was stable to acid and basic hydrolysis and thermolysis but not to oxidation. POS and its degradation products were characterized by high-resolution positive mode electrospray ionization quadrupole time-of-flight (ESI-QTOF) mass spectrometry. Two degradation products with m/z values of 717 and 733 were detected. Molecular modeling was used for the calculation of the highest occupied molecular orbital (HOMO) and electrostatic potential changes. The results suggest that POS degradation begins with the breakage of its piperazine ring. This is the first study to report the kinetics of POS oxidative degradation under different oxidizing agent concentrations, temperatures, and pH conditions. The reaction followed second-order kinetics under all tested conditions. The analytical methods developed in this study were suitable to delineate the stability profile of POS. Elucidation of the degradation kinetics of POS is essential to ensure the stability of the drug in pharmaceutical formulations.

Chemical, structural and functional properties of whey proteins covalently modified with phytochemical compounds

This work was conducted to understand the covalent interactions of whey protein isolate (WPI) with some phytochemical compounds including chlorogenic acid (CQA), rosmarinic acid (RA) and quercetin (Q) (0.3 mmol/g protein). The influences of such interactions on the chemical, structural and functional properties of the WPI were studied. The changes in protein–phenolic conjugates properties were characterized by the change in free amino and thiol groups, tryptophan content, UV–Vis scan and particle size. Moreover, the electrospray ionization-quadrupole-time of flight (ESI-Q-TOF) mass spectrometry, RP-HPLC, TEAC and DPPH assays were also used to evaluate these changes. In addition, the change in isoelectric points of modified proteins was examined using the change in the zeta potential at different pH values. The results showed that these interactions caused a decrease in both free amino and thiol groups and tryptophan content, where the addition of CQA, RA and Q led to decrease in the thiol groups content by about 47.6, 96.1 and 65.8%, respectively. While, the content of tryptophan residue decreased from 29.9 ± 0.3 (nmol/mg protein, for WPI control) to 19.3 ± 0.1, 7.9 ± 0.1 and 17.2 ± 1.0 after interacting with CQA, RA and Q, respectively. The structure of WPI- phenolic conjugates altered compared to the structure of WPI control. The modified proteins displayed very slight shifts in the isoelectric points compared to the unmodified ones. Moreover, these interactions increased the antioxidant capacity of proteins about 2.7 to 3.4 folds compared to WPI control. Therefore, it is strongly recommended to use the modified WPI for preparing functional food products with a high antioxidative power.

Photodegradation Study of Sertindole by UHPLC-ESI-Q-TOF and Influence of Some Metal Oxide Excipients on the Degradation Process.

The evaluation of the influence of the excipients present in the pharmaceutical formulations on the drug stability is an important part of quality control of medicines. One of the most commonly applied group of excipients are pigments, such as titanium dioxide or various forms of iron oxides, which are well-known photocatalytic agents. Therefore, the photostability of an atypical antipsychotic drug sertindole and the influence of pigments commonly used in the pharmaceutical formulations (FeOOH, Fe2O3, and TiO2) on this process were studied. The quantitative and qualitative analysis of the process was performed with the use of ultra high pressure liquid chromatography with diode array detection (UHPLC-DAD) system coupled with a high resolution hybrid electrospray ionization quadrupole time-of-flight (ESI-Q-TOF) mass spectrometer. Sertindole turned out to be a highly photolabile molecule. Overall 18 transformation products were found, mainly formed as a consequence of dechlorination, hydroxylation, and dehydrogenation. In all the experiments, except the TiO2-mediated photocatalysis, the product of chlorine substitution with a hydroxyl group was the major product. The presence of Fe2O3 and TiO2 accelerated the degradation process, while FeOOH served as its inhibitor. The experiments conducted with the use of the pharmaceutical formulations confirmed the catalytic activity of the used excipients. The exploration of the obtained phototransformation profiles with the use of principal component analysis (PCA) revealed that the presence of both iron oxides could influence the qualitative and quantitative aspect of the studied processes. In silico assessment of the properties showed that the transformation products are generally less toxic to rodents, possess lower hERG blocking potential, but could be more mutagenic than the parent molecule.

Spectroscopic Characterization and Cytotoxicity Assessment towards Human Colon Cancer Cell Lines of Acylated Cycloartane Glycosides from Astragalus boeticus L.

In several European countries, especially in Sweden, the seeds of the species Astragalus boeticus L. were widely used as coffee substitutes during the 19th century. Nonetheless, data regarding the phytochemistry and the pharmacological properties of this species are currently extremely limited. Conversely, other species belonging to the Astragalus genus have already been extensively investigated, as they were used for millennia for treating various diseases, including cancer. The current work was addressed to characterize cycloartane glycosides from A. boeticus, and to evaluate their cytotoxicity towards human colorectal cancer (CRC) cell lines. The isolation of the metabolites was performed by using different chromatographic techniques, while their chemical structures were elucidated by nuclear magnetic resonance (NMR) (1D and 2D techniques) and electrospray-ionization quadrupole time-of-flight (ESI-QTOF) mass spectrometry. The cytotoxic assessment was performed in vitro by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays in Caco-2, HT-29 and HCT-116 CRC cells. As a result, the targeted phytochemical study of A. boeticus enabled the isolation of three new cycloartane glycosides, 6-O-acetyl-3-O-(4-O-malonyl)-β-d-xylopyranosylcycloastragenol (1), 3-O-(4-O-malonyl)-β-d-xylopyranosylcycloastragenol (2), 6-O-acetyl-25-O-β-d-glucopyranosyl-3-O-β-d-xylopyranosylcycloastragenol (3) along with two known compounds, 6-O-acetyl-3-O-β-d-xylopyranosylcycloastragenol (4) and 3-O-β-d-xylopyranosylcycloastragenol (5). Importantly, this work demonstrated that the acetylated cycloartane glycosides 1 and 4 might preferentially inhibit cell growth in the CRC cell model resistant to epidermal growth factor receptor (EGFR) inhibitors.

Interactions between human hemoglobin subunits and peroxiredoxin 2.

Red blood cells (RBCs) are exposed to exogenous reactive oxygen species in the circulatory system. To this end, the interactions between the different hemoglobin (Hb) subunits and peroxiredoxin 2, which is a ubiquitous member of the antioxidant enzymes that also controls the cytokine-induced peroxide levels, were assessed. We predicted by the increment of diversity with quadratic discriminant analysis (IDQD) that peroxiredoxin2 (Prx2) could interact with the hemoglobin alpha, beta and gamma subunits but not with the delta subunit. Coimmunoprecipitation (co-IP), electrosprayionization quadrupole timeof flight (ESI-Q-TOF) mass spectrometry, Western blotting and X-ray absorption fine structure (XAFS) spectroscopy were performed to verify these predictions. The results showed that Prx2 was a member of the beta-globin immunoprecipitating complex that existed in hemoglobin A, hemolysate-hemoglobin A, hemoglobin A-hemoglobin A2, hemolysate-hemoglobin A-hemoglobin A2 and hemoglobin A2 but not in hemolysate-hemoglobin A2. Adding Prx2 to hemoglobin A altered the second shell of iron embedded in hemoglobin A. Therefore, Prx2 interacts with hemoglobin A (Alpha2Beta2) and hemoglobin F (Alpha2Gamma2) but not with hemoglobin A2 (Alpha2Delta2).

Selection of Collision Energies in Proteomics Mass Spectrometry Experiments for Best Peptide Identification: Study of Mascot Score Energy Dependence Reveals Double Optimum.

Collision energy is a key parameter determining the information content of beam-type collision induced dissociation tandem mass spectrometry (MS/MS) spectra, and its optimal choice largely affects successful peptide and protein identification in MS-based proteomics. For an MS/MS spectrum, quality of peptide match based on sequence database search, often characterized in terms of a single score, is a complex function of spectrum characteristics, and its collision energy dependence has remained largely unexplored. We carried out electrospray ionization-quadrupole-time of flight (ESI-Q-TOF)-MS/MS measurements on 2807 peptides from tryptic digests of HeLa and E. coli at 21 different collision energies. Agglomerative clustering of the resulting Mascot score versus energy curves revealed that only few of them display a single, well-defined maximum; rather, they feature either a broad plateau or two clear peaks. Nonlinear least-squares fitting of one or two Gaussian functions allowed the characteristic energies to be determined. We found that the double peaks and the plateaus in Mascot score can be associated with the different energy dependence of b- and y-type fragment ion intensities. We determined that the energies for optimum Mascot scores follow separate linear trends for the unimodal and bimodal cases with rather large residual variance even after differences in proton mobility are taken into account. This leaves room for experiment optimization and points to the possible influence of further factors beyond m/ z.

Application of an ESI-QTOF method for the detailed characterization of GSK-3β inhibitors

The crucial role of Glycogen Synthase Kinase 3 (GSK-3β) as a pivotal player in Alzheimer's Disease (AD) has recently inspired significant attempts to design and synthesize potent kinase inhibitors. In fact GSK-3β is considered the main kinase which catalyzes the microtubule-associated protein tau hyper-phosphorylation and the neurofibrillary tangles (NFT) in vitro and in vivo, The first classes of GSK-3β inhibitors were classified as ATP-competitive and, therefore, they lack of an efficient degree of selectivity over other kinases. In light of this consideration, many efforts are devoted to characterize new non ATP-competitive GSK-3β inhibitors, endowed with high selectivity. In parallel, there is an urgent need to develop new analytical methodologies for the hit selection (highthroughput screening) and ligand binding characterization in terms of potency, affinity and mechanism of action. The new methodology for GSK-3β enzymatic activity determination can be adopted as a realistic alternative to the currently used radioactive, luminescence and fluorescence detection methods, each showing limitations in terms of safety and interferences. Herein, we propose an alternative and selective electrospray ionization quadrupole time-of-flight (ESI-QTOF) method, based on the direct quantification of phosphorylated substrate muscle glycogen synthase GSM, a peptide resembling the high affinity sequence of natural substrate muscle glycogen synthase 1, for the detailed characterization of GSK-3β inhibitors. The method was validated in terms of accuracy and reproducibility of GSM signal intensity with a relative standard deviation RSD% value of 3.55%; Limit of Detection (LOD): 0.006μM; Lower Limit of Quantification (LLOQ): 0.02μM; linearity r2 0.9951. The kinetic constants (KM and vmax) of the GSK-3β catalyzed kinase reaction and the inhibitory potency of known ligands (IC50), were determined. All the obtained results were in agreement with those reported in literature or obtained in house by the standard reference luminometric approach. The proposed method was applied to the elucidation of well known inhibitors mechanism of action by the construction of a Lineweaver–Burk plot and the Ki determination. Furthermore, the potency, affinity and mechanism of action of a new non ATP-competitive compound were established. We demonstrated the ESI-QTOF method to be more feasible than the classic kinase assays since it avoids drawbacks inherently connected with radioisotope labeling or the indirect detection of kinase activity, so far. It is also scalable to the screening of large library collections and suitable for pharmaceutical industries purposes.

Monitoring protein glycation by electrospray ionization (ESI) quadrupole time-of-flight (Q-TOF) mass spectrometer

In this study electrospray ionization quadrupole time-of-flight (ESI-Q-TOF) mass spectrometry was used to investigate protein glycation. The glycated species of cytochrome C, lysozyme, and β-casein formed during glycation with d-glucose were identified and monitored in binary systems heated at 70°C under dry and aqueous conditions. Cytochrome C had multiple charges in non-glycated state, primarily changing from +13 to +17 positive charges, whereas β-casein had charge states up to +30. Upon heating with glucose at 70°C in aqueous state, attachment of one glucose molecule onto proteins was observed in each charge state. However, heating in dry state caused much more glucose attachment, leading to the formation of multiple glycoforms of proteins. By using ESI-QTOF-MS technique, formation of glycated cytochrome C containing up to 12 glucose moieties were observed, while glycated species containing 6 and 8 glucose moieties were observed for lysozyme and β-casein, respectively in various heating conditions.

Two competitive INC-mediated reactions in the gas-phase fragmentation of protonated indolyl benzo[b]carbazoles.

Ion-neutral complexes (INCs) are intermediates extensively existing in gas-phase ionic reactions. Tandem mass spectrometry (MS(n) ) analysis of indolyl benzo[b]carbazoles was performed on an electrospray ionization quadrupole time-of-flight (ESI-Q-TOF) mass spectrometer in positive ion mode. Two competing INC-mediated reactions were obtained in the fragmentation of protonated indolyl benzo[b]-carbazoles. This study enriches our knowledge of the important roles of INCs in the dissociation of indole compounds in the gas phase. Copyright © 2016 John Wiley & Sons, Ltd.

Quadruple-stranded Eu-helicate assembled from bis-β-diketonate: Its stability towards metal ions

Herein, we reported the synthesis and investigation of highly luminescent quadruple-stranded helicate (C6H16N)4[Eu2(MBDA)4]2 ·3C4H10O·4C2H3N(1-Eu)[H2MBDA=N-methyl-4,4′-bis(4,4,4-trifluoro-1,3-dioxobutyl)diphenylamine] for its stability toward metal ions in the solution. The material was characterized via X-ray crystallographic technique, Fourier transform infrared(FTIR) spectroscopy and electrospray ionization quadrupole time-offlight(ESI-TOF) mass spectrometry. The results on the luminescence quantum yields clearly demonstrate that the ligand can effectively sensitize the luminescence of the Eu3+ ions(Φ overall=15%). Upon the addition of different metal ions(i. e., Ag+, Cd2+, Zn2+, Fe3+, Al3+ and Ni2+) to the CH3CN solution of compound 1-Eu, the emission intensities of Eu3+ ions at 612 nm were affected to some extent, which could be attributed to the presence of ion exchanges between Eu3+ ions and the metals ions, and the result was confirmed by ESI-TOF mass spectrometry.

Gas-phase fragmentation of the N-oxide and N-hydroxylated derivatives of retrorsine using liquid chromatography/electrospray ionization quadrupole time-of-flight tandem mass spectrometry.

We report the electrospray ionization mass spectrometry and low-energy collision-induced dissociation tandem mass spectrometry (CID-MS/MS) analysis of a pyrrolizidine alkaloid extract containing both retrorsine [C18H25NO6] and its N-oxide [C18H25NO7] and N-hydroxyl [C18H26NO7] derivatives measured with a QqTOFMS hybrid instrument. A solution of the pyrrolizidine alkaloid extract containing retrorsine and its N-oxide and N-hydroxyl derivatives was directly infused into an electrospray ionization-quadrupole-time-of-flight (ESI-QTOF) mass spectrometer and product ion scans of the protonated molecules of each species were acquired. Labile protons of each compound were deuterated and computational energy calculations of the proposed structures of the product ions were used to determine the fragmentation pathways of retrorsine and its N-oxide and N-hydroxyl derivatives. ESI-MS of the pyrrolizidine alkaloid extract containing retrorsine and its N-oxide and N-hydroxyl derivatives afforded the protonated retrorsine [M1 + H](+) at m/z 352.1760 and the protonated retrorsine N-oxide [M2 + H](+) at m/z 368.1631 in addition to the formation of the unexpected protonated N-hydroxyl radical [M3 + H](+•) at m/z 369.1686. CID-MS/MS of this series of protonated molecules allowed the evaluation of their gas-phase fragmentations and the establishment of their fragmentation pathways. It was also found that several product ions could be assigned to different structures. Deuterium exchange and computational energy calculations allowed us to determine the most probable structures for the characterized product ions. To our knowledge, the identification of the protonated retrorsine N-hydroxyl radical [M3 + H](+•) is reported for the first time. In addition, the MS/MS results can be used for the identification of retrorsine and its N-oxide and N-hydroxyl derivatives in different complex biological matrices.

New evidence for H/D scrambling of tryptophan and its analogues in the gas phase

Tryptophan and its analogues were analyzed using electrospray ionization quadrupole-time of flight (ESI-QTOF) combined with hydrogen–deuterium exchange. The similar profiles of MS3 spectra of the product ions at m/z 190, 191, and 192 from [M (D)+D]+ for tryptophan by the losses of NH(D)3 imply the same fragmentation patterns including the characteristic transfer of hydroxyl and very similar structures of these ions. This reveals the H/D scrambling prior to fragmentation. Compared the MS/MS spectrum of D-labelled compound 2 to the MS3 spectrum of m/z 191, it was found that amino group plays an important role for the H/D scrambling. The MS2 and MS3 spectra for D-labelled compound 4 indicate that the H/D scrambling involves the hydrogen on methyl linked to indole ring. In addition, the fragmentation of transfer of hydroxyl or methoxyl is confirmed by comparing the spectra of compounds 1–4.

Capillary electrophoresis-mass spectrometry determination of morphine and its isobaric glucuronide metabolites.

The determination of morphine and its isobaric metabolites morphine-3-beta-d-glucuronide (M3G) and morphine-6-beta-d-glucuronide (M6G) is useful for therapeutic drug monitoring and forensic identification of drug use. In particular, capillary electrophoresis with mass spectrometry (CE-MS) represents an attractive tool for opioid analysis. Whereas volatile background electrolytes in CE often improve electrospray ionization for coupled MS detection, such electrolytes may reduce CE separation efficiency and resolution. To better understand the effects of background electrolyte (BGE) composition on separation efficiency and detection sensitivity, this work compares and contrasts method development for both volatile (ammonium formate and acetate) and nonvolatile (ammonium phosphate and borate) buffers. Peak efficiencies and migration times for morphine and morphine metabolites were optimal with a 25mM ammonium borate buffer (pH=9.5) although greater sensitivities were achieved in the ammonium formate buffer. Optimized CE methods allowed for the resolution of the isobaric morphine metabolites prior to high mass accuracy, electrospray ionization quadrupole time-of-flight (ESI-QTOF) MS detection applicable to the analysis of urine samples in under seven minutes. Urine sample preparation required only a 10-fold dilution with BGE prior to analysis. Limits of detection (LOD) in normal human urine were found to be 1.0μg/mL for morphine and 2.5μg/mL for each of M3G and M6G by CE-ESI-QTOF-MS. These LODs were comparable to those for CE-UV analysis of opioid standards in buffer, whereas CE-ESI-QTOF-MS analysis of opioid standards in buffer yielded LODs an order of magnitude lower. Patient urine samples (N=12) were analyzed by this new CE-ESI-QTOF-MS method and no significant difference in total morphine content relative to prior liquid chromatography-mass spectrometry (LC-MS) results was found as per a paired-t test at the 99% confidence level. Whereas the LC-MS method applied to these samples determined only total morphine content, this new CE-ESI-QTOF-MS method allowed for species differentiation in addition to total morphine determination. By this method, it was found that M3G and M6G metabolites were present in a 5:1 concentration ratio, on average, in patient samples. Therefore, the CE-ESI-QTOF-MS method not only allows for total morphine concentration determination comparable to established LC-MS methods, but also allows for differentiation between morphine and its trace glucuronides, yielding additional biochemical information about drug metabolism.

Transformation of gas-phase amino acid clusters to dipeptides: a nice approach to demonstrate the formation of prebiotic peptides.

Exploring prebiotic developments is a fascinating area of research which is continually drawing the attention of the scientific community. It is probable that first the biomolecules were formed and then they became aggregated to generate life. Formation of one such biomolecules (peptide ions) is shown in the present experiments. All amino acid solutions for recording mass spectra were prepared in 3:6.9:0.1 (v/v/v) acetonitrile/water/formic acid at a concentration of 50 μM. The studies were performed using a Bruker MicroTOF QII mass spectrometer. Before carrying out experiments in the collision cell, atmospheric pressure in-source fragmentations were also performed. The formation of different chemical species was detected with high-resolution mass spectrometry. Here, we show experimentally the formation of amino acid cluster ions of varied populations, when a solution of an amino acid was injected into an electrospray ionization quadrupole time-of-flight (ESI-QTOF) mass spectrometer. During in-source fragmentation/collision cell fragmentation, the non-covalent interaction between two identical amino acids forms either the [M2 + H](+) dimer cluster ion and/or the [M2 + K](+) adduct ion which, by elimination of one molecule of water, form the covalent linked dipeptide. After the formation of the amino acid cluster, it was established that the creation of the dipeptides, by a covalent bond resulting from the loss of a water molecule, was the initial step towards the formation of the primordial peptides.

Identification of triacylglycerols in archaelogical organic residues by core–shell reversed phase liquid chromatography coupled to electrospray ionization-quadrupole-time of flight mass spectrometry

A method based on the use of a Poroshell 120 EC-C18 column (3.0mm×50mm, 2.7μm) with a high resolution electrospray ionization-quadrupole-time of flight (ESI-Q-ToF) tandem mass spectrometer as detection system was optimized for the identification of triacylglycerols (TAGs) in complex archaeological residues.The best performances in chromatographic separation and Q-ToF detection sensitivity were reached by using a mixture of iPrOH/MeOH at a 0.6mL/min flow rate. Triolein (OOO) was eluted in 12.30min and the chromatographic resolution against tripalmitin (PPP) was 1.4. Very good limits of detection and quantification limits were also achieved (LOD<0.03μg/g LOQ<0.10μg/g).These achievements were compared with the results provided by traditional particle columns and with the results reported in the recent literature regarding TAGs analysis in archaeological residues. Thanks to the unprecedented chromatographic separation and detection sensitivity attained, it was possible for the first time to perform TAGs researches in archaeological residues and reference materials with a dataset containing more than 500 molecular formula.