Electrospray Ionization Quadrupole Time-of-flight Mass Spectrometry Research Articles

Analytical method development, identification, and characterization of stress degradation products of idelalisib by ultrahigh-performance liquid chromatography with photodiode array and ultrahigh-performance liquid chromatography with electrospray ionization quadrupole time-of-flight mass spectrometry studies.

As per International Council for Harmonization (ICH) drug stability test guideline Q1A(R2), inherent stability characteristics of a drug should be studied. This work was designed to investigate inherent degradation characteristics of the drug idelalisib under ICH prescribed stress conditions, identify its degradation products, and postulate their corresponding degradation pathways. Idelalisib was subjected to the ICH prescribed conditions of hydrolytic (neutral, acidic, and alkaline), photolytic, oxidative, and thermal stress according to ICH guideline Q1A(R2). An ultrahigh-performance liquid chromatography with photodiode array (UHPLC-PDA) method was developed to adequately resolve the drug from its degradation products, validated as per the ICH guidelines, and subsequently extended to UHPLC with electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-QTOFMS) studies to identify the degradation products. Significant degradation was noted under conditions of acidic/alkaline hydrolysis, acid photolysis, and oxidative stress. The UHPLC/ESI-QTOFMS studies revealed the generation of four degradation products (I-IV), which were satisfactorily resolved from the drug by UHPLC on a Kinetex® C18 (100 × 4.6mm; 2.6μm) column by the developed isocratic elution method. Detection wavelength was selected as 270 nm. All the degradation products (I-IV) could be identified and characterized from their mass spectral data. The degradation pathways for the generation of various products from the drug were postulated. A UHPLC-PDA method was developed and validated for idelalisib. Four degradation products of idelalisib were revealed through UHPLC/ESI-QTOFMS studies, and corresponding degradation pathways were postulated for the same.

High NaCl concentrations induce the resistance to thermal denaturation of an extremely halotolerant (salt-activated) β-mannanase from Bacillus velezensis H1.

β-mannanase catalyzes the hydrolysis of mannans β-1,4-mannosidic linkages to produce industrially relevant oligosaccharides. These enzymes have numerous important applications in the detergent, food, and feed industries, particularly those that are resistant to harsh environmental conditions such as salts and heat. While, moderately salt-tolerant β-mannanases are already reported, existence of a high halotolerant β-mannanase is still elusive. This study aims to report the first purification and characterization of ManH1, an extremely halotolerant β-mannanase from the halotolerant B. velezensis strain H1. Electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOF-MS) analysis revealed a single major peak with a molecular mass of 37.8kDa demonstrating its purity. The purified enzyme showed a good thermostability as no activity was lost after a 48h incubation under optimal conditions of 50°C and pH 5.5. The enzyme's salt activation nature was revealed when its maximum activity was obtained in the presence of 4M NaCl,it doubled compared to the no-salt condition. Moreover, NaCl strengthens its resistance to thermal denaturation, as its melting temperature (Tm) increased steadily with increasing NaCl concentrations reaching 75.5°C in the presence of 2.5M NaCl. The Km and Vmax values were 5.63mg/mL and 333.33 µmol/min/mL, respectively, using carob galactomannan (CG) as a substrate. The enzyme showed a significant ability to produce manno-oligosaccharides (MOS) from lignocellulosic biomass releasing 13mg/mL of reducing sugars from olive mill wastes (OMW)after 24h incubation. The results revealed that this enzyme may have significant commercial values for agro-waste treatment, and other potential applications.

Untargeted Metabolomic Approach of Curcuma longa to Neurodegenerative Phytocarrier System Based on Silver Nanoparticles.

Curcuma is one of the most famous medicinal and tropical aromatic plants. Its health benefits have been appreciated and exploited in traditional Asian medicine since ancient times. Various studies have investigated its complex chemical composition and demonstrated the remarkable therapeutic properties of curcuma's phytoconstituents. Oxidative stress is a decisive driving factor triggering numerous pathologies (neurodegenerative, psychiatric and cardiovascular diseases; diabetes; tumors, etc.). Numerous recent studies have focused on the use of natural compounds and nanomaterials as innovative molecular targeting agents as effective therapeutic strategies. In this study, we report, for the first time, the development of a simple target phytocarrier system that capitalizes on the bioactive properties of curcuma and AgNPs. The complete metabolic profile of curcuma was determined based on gas chromatography-mass spectrometry (GC-MS) and electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-QTOF-MS). A total of 80 metabolites were identified under mass spectra (MS)-positive mode from 10 secondary metabolite categories: terpenoids, amino acids, diarylheptanoids, flavonoids, phenolic acids, steroids, fatty acids, coumarins, alkaloids and miscellaneous. In addition, the biological activity of each class of metabolites was discussed. A comprehensive characterization (FT-IR, UV-Vis, DLS, SEM, TEM, EDS, zeta potential and XRD) was performed to study the morphostructural properties of this new phytocarrier system. Antioxidant activity of the new phytocarrier system was evaluated using a combination of in vitro methods (total phenolic assay, 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay and cyclic voltammetric method (Trolox equivalent antioxidant capacity (TEAC) electrochemical assay)). Antioxidants assays showed that the phytocarrier system exhibits superior antioxidant properties to those of its components, i.e., curcuma or citrate-coated-AgNPs. These data confirm the potential to enhance relevant theoretical knowledge in the area of innovative antioxidant agents, with potential application in neurodegenerative therapeutic strategies.

Secondary Metabolite Profile and Pharmacological Opportunities of Lettuce Plants following Selenium and Sulfur Enhancement.

Selenium (Se) is an essential trace nutrient for humans and animals owing to its role in redox regulation, thyroid hormone control factors, immunity, inflammatory reactions, brain activities, and carbohydrate regulation. It is also important to support muscle development, as well as for reproductive and cardiovascular well-being. Furthermore, sulfur is known to be a healing element, due to the remarkable function of specialized and secondary S-containing compounds. The scope of the current study was to determine the impact of Se and S enrichment on the secondary metabolite accumulation and antibacterial and NO inhibition activities in green and red leaf lettuce (V1 and V2, respectively). The plants were grown in a hydroponic system supplied with different S concentrations (S0: 0, S1: 1 mM and S2: 1.5 mM K2SO4) via the nutrient solution and foliar-applied varying levels of Se (0, 0.2 and 2.6 µM). Electrospray ionization-quadrupole time-of-flight mass spectrometry (ESI-QTOF/MS) combined with ultra-performance liquid chromatography (UPLC) was used to identify the secondary metabolites in green and red lettuce. The results indicated that extracts of the biofortified lettuce were not cytotoxic to Vero kidney cells at the highest concentration tested of 1 mg/mL. The ESI/MS of the tentatively identified metabolites showed that the response values of 5-O-caffeoylquinic acid, cyanidin 3-O-galactoside, quercetin 3-O-(6''-acetyl-glucoside) and quercetin 3-O-malonylglucoside were induced synergistically under higher Se and S levels in red lettuce plants. The acetone extract of red lettuce had antibacterial activity against Pseudomonas aeruginosa, with a minimum inhibitory concentration (MIC) of 0.156 and 0.625 μg/mL under S2/Se1 and S2/Se2 treatments, respectively. As with antibacterial activity, the acetone extract of green (V1) lettuce treated with adequate (S1) and higher S (S2) under Se-limiting conditions showed the ability to inhibit nitric oxide (NO) release from macrophages. NO production by macrophages was inhibited by 50% at respective concentrations of 106.1 ± 2.4 and 101.0 ± 0.6 μg/mL with no toxic effect on the cells, in response to S1 and S2, respectively, under Se-deficient conditions (Se0). Furthermore, the red cultivar (V2) exhibited the same effect as the green cultivar (V1) regarding NO inhibition, with IC50 = 113.0 ± 4.2 μg/mL, in response to S1/Se2 treatments. Collectively, the promising NO inhibitory effect and antibacterial activity of red lettuce under the above-mentioned conditions might be attributed to the production of flavonoid glycosides and phenylpropanoic acid esters under the same condition. To the best of our knowledge, this is the first report to show the novel approach of the NO inhibitory effect of Se and S enrichment in food crops, as an indicator for the potential of Se and S as natural anti-inflammatory agents.

Identification of cadmium containing metabolites in HepG2 cells after treatment with cadmium-selenium quantum dots

The transformation of quantum dots (QDs) by organisms has attracted broad attention but remains unclear. Understanding of the metabolites helps to reveal the transformation pathway of QDs. Cd containing-metallothionein (MT) are the main species formed by Cd released from CdSe QDs in HepG2 cells, while speciation analysis of Cd containing MTs remains a challenge because MTs has several subisoforms and can bind with several metals. Herein, we built a hyphenated platform for speciation analysis of QDs in HepG2 cells after treatment with CdSe/ZnS QDs. The Cd-containing MTs were separated in reversed phase high performance liquid chromatography (RP-HPLC) and subsequently online detected by inductively coupled plasma mass spectrometry (ICP-MS) and electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOF-MS) parallelly. Four groups of Cd-containing metabolites were found by detecting Cd in ICP-MS. Their structures were identified in ESI-Q-TOF-MS and further confirmed with standards of four subisoforms of MT, including N-terminal acetylation MT2a, N-terminal acetylation MT1e, N-terminal acetylation MT1g and MT1m. Each group of them contains various stoichiometry of Cd/Zn. The metabolites of QDs remain same while the concentrations of each metabolite and its stoichiometry of Cd/Zn vary for different incubation concentration/time. This work provides a new parallel hyphenation technique of HPLC-ICP-MS/ESI-MS with high separation resolution and powerful detection ability, and the obtained results provide detailed metabolism information of QDs in HepG2 cells after treatment of CdSe/ZnS QDs, contributing to deep exploration of the functional mechanisms of QDs in organisms.

Multimeric Association of Purified Novel Bowman-Birk Inhibitor From the Medicinal Forage Legume Mucuna pruriens (L.) DC.

A Bowman-Birk protease, i.e., Mucuna pruriens trypsin inhibitor (MPTI), was purified from the seeds by 55.702-fold and revealed a single trypsin inhibitor on a zymogram with a specific activity of 202.31 TIU/mg of protein. On sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) under non-reducing conditions, the protease trypsin inhibitor fraction [i.e., trypsin inhibitor non-reducing (TINR)] exhibited molecular weights of 74 and 37 kDa, and under reducing conditions [i.e., trypsin inhibitor reducing (TIR)], 37 and 18 kDa. TINR-37 revealed protease inhibitor activity on native PAGE and 37 and 18 kDa protein bands on SDS–PAGE. TINR-74 showed peaks corresponding to 18.695, 37.39, 56.085, and 74.78 kDa on ultra-performance liquid chromatography (UPLC) coupled with electrospray ionization/quadrupole time-of-flight-mass spectrometry (ESI/QTOF-MS). Similarly, TINR-37 displayed 18.695 and 37.39 kDa peaks. Furthermore, TIR-37 and TIR-18 exhibited peaks corresponding to 37.39 and 18.695 kDa. Multiple peaks observed by the UPLC-ESI/QTOF analysis revealed the multimeric association, confirming the characteristic and functional features of Bowman-Birk inhibitors (BBIs). The multimeric association helps to achieve more stability, thus enhancing their functional efficiency. MPTI was found to be a competitive inhibitor which again suggested that it belongs to the BBI family of inhibitors, displayed an inhibitor constant of 1.3 × 10–6 M, and further demonstrates potent anti-inflammatory activity. The study provided a comprehensive basis for the identification of multimeric associates and their therapeutic potential, which could elaborate the stability and functional efficiency of the MPTI in the native state from M. pruriens.

Impurity profiling of siRNA by two-dimensional liquid chromatography-mass spectrometry with quinine carbamate anion-exchanger and ion-pair reversed-phase chromatography

A short RNA with the sequence of the antisense strand of Patisiran has been selected as test material for the investigation of its common impurities using three different two-dimensional liquid chromatography (2D-LC) platforms. On the one hand, a quinine (QN) carbamate-based weak anion-exchange (AX) stationary phase (QN-AX) and a classical C18 reversed phase (RP) stationary phase in ion-pair (IP) mode with tripropylammonium acetate, respectively, have been used in the first dimension (1D) to provide the selectivity for impurities formed during the synthesis of the RNA. In the next step, certain peaks of interest from 1D have been transferred by multiple-heart-cutting (MHC) into a 2D in which an ESI-MS-compatible non-ionpairing RP method has been used for desalting via a diverter valve to remove non-volatile phosphate buffer components and ion-pair agents, respectively. Thus, a sensitive electrospray-ionization quadrupole time of flight mass spectrometry (ESI-TOF-MS) analysis of resolved impurity peaks of the siRNA has become possible under MS-friendly conditions. With both 2D-LC setups, peak purity of the ON has been evaluated by selective comprehensive (high resolution) sampling of the main peak. In a third MHC 2D-LC approach, the QN-AX LC mode was online coupled with the IP-RPLC in the 2D using UV detection. It allows the separation of additional impurities which coeluted in the first dimension. The potential of these methods for comprehensive impurity profiling of ON therapeutics is illustrated and discussed.

Chemical Screening of Metabolites Profile from Romanian Tuber spp.

Truffles are the rarest species and appreciated species of edible fungi and are well-known for their distinctive aroma and high nutrient content. However, their chemical composition largely depends on the particularities of their grown environment. Recently, various studies investigate the phytoconstituents content of different species of truffles. However, this research is still very limited for Romanian truffles. This study reports the first complete metabolites profiles identification based on gas chromatography-mass spectrometry (GC-MS) and electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-QTOF-MS) of two different types of Romania truffles: Tuber magnatum pico and Tuber brumale. In mass spectra (MS) in positive mode, over 100 metabolites were identified from 14 secondary metabolites categories: amino acids, terpenes, alkaloids, flavonoids, organic acids, fatty acids, phenolic acids, sulfur compounds, sterols, hydrocarbons, etc. Additionally, the biological activity of these secondary metabolite classes was discussed.

Cadmium species in marine short-necked clam analyzed by size exclusion chromatography hyphenated respectively with inductively coupled plasma mass spectrometry and electrospray ionization quadrupole - time of flight mass spectrometry

To date, the speciation analysis of heavy metals in organs is still a challenge because these species are labile in vitro and scarce of the reference materials. In the present study, cadmium (Cd) species in marine shellfish were investigated with size exclusion chromatography (SEC) hyphenated with inductively coupled plasma mass spectrometry (ICP-MS), by which three Cd species in marine short-necked clam were observed. After pre-fractionation with a home-made Sephadex column, the Cd binding species were further characterized with SEC coupled with electrospray ionization quadrupole - time of flight mass spectrometry (ESI-Q-TOF-MS). The results showed that Cd species with 32,695 Da was the maximum Cd-binding molecule in the clam. Several metallothionein (MTs)-like Cd-binding proteins including a main species with molecular weight of 19,236 Da were observed. Some small Cd species with molecular weights less than 1000 Da were also observed in the clam. In order to verify the reliability of the two hyphenated methods, a standard reference material of Zn-binding MTs was analyzed at the same conditions. The results demonstrated that the two hyphenated methods of SEC-ICP-MS and SEC-ESI-Q-TOF-MS were practical to characterize the Cd species in marine shellfish.

Rapid Determination of Furoquinoline Alkaloids in Rutaceae Species by Ultra-Performance Liquid Chromatography (UPLC) with Photodiode Array (PDA) and Electrospray Ionization–Quadrupole Time-of-Flight Mass Spectrometry (ESI-Q-TOF/MS)

A qualitative and quantitative method based on ultra-performance liquid chromatography (UPLC) and photodiode array (PDA) and electrospray ionization–quadrupole time-of-flight mass spectrometry (ESI-Q-TOF/MS) was developed, verified, and used to determine the quinoline alkaloids in Dictamnus dasycarpus and Zanthoxylum schinifolium, belonging to the Rutaceae family. Excellent analyte peak resolution was achieved on both quaternary and binary pumps by using acetonitrile and water containing 0.1% formic acid as the mobile phase using a C18 column. All alkaloids exhibited maximum absorption wavelength from 239 to 244 nm. The analytes were ionized as quasi-molecular ions [M + H]+ in MS and were characterized by using fragmentation ions formed by collision-induced dissociation (CID). This information was included in a library that was used to qualitatively identify alkaloids. The proposed method showed excellent linearity (r2 > 0.9988), limits of detection (PDA ≤ 25 ng/mL, MS ≤ 1.2 ng/mL), limits of quantification (PDA ≤ 75 ng/mL, MS ≤ 3.6 ng/mL), precision (in terms of relative standard deviation) of PDA ≤ 1.81% and MS 5.7%,. Liquid–liquid extraction (LLE) and solid-phase extraction (SPE) were conducted to separate and purify the alkaloids from the extracts. The analyte recoveries were determined to be PDA ≤ 103.11% and MS ≤ 104.45%. The method proceeded to deliver the comparable results for the determination of alkaloid components in samples. The developed UPLC-PDA and UPLC-ESI-Q-TOF/MS protocol facilitated the rapid, qualitative and quantitative identification of alkaloids in plants.

Untargeted metabolite profiling and phytochemical analysis based on RP-HPLC-DAD-QTOF-MS and MS/MS for discovering new bioactive compounds in Rumex algeriensis flowers and stems.

Natural products with distinctive pharmaceutical activities are considered as the main source of new herbal drugs, functional foods and cosmetic additives. Rumex algeriensis is an endemic medicinal plant with no accessible information about its chemical profile and biological activities. In the quest for new sources of biologically-active compounds, we intended in the present work to undertake a comprehensive characterisation of phytochemical compounds from Rumex algeriensis flowers and stems hydro-methanolic extract. Chemical profiles were evaluated by a rapid analytical method reversed-phase high-performance liquid chromatography (RP-HPLC) coupled to electrospray ionisation-quadrupole-time-of-flight mass spectrometry (ESI-QTOF-MS) and tandem mass spectrometry (MS/MS) using negative and positive ions modes. In this work, 44 bioactive compounds were tentatively identified using high mass accuracy data and confirmed by MS/MS experiments, among which five compounds were reported for the first time in the Polygonaceae family. These compounds were classified as sugars, hydroxybenzoic acids, hydroxycinnamic acids, flavonols, flavanones, flavone, flavanols, condensed tannins, hydrolysable tannins and their conjugated derivatives. The obtained results highlighted that Rumex algeriensis, even though undervalued and unexplored, contributes to the preclinical knowledge that could be considered as a renewable source of nutraceutical compounds that may be exploited in novel herbal medicinal products, in the food, pharmaceutical and nutraceutical industries.

Proteomic Profile of Carbonylated Proteins Screen the Regulation of Calmodulin-Dependent Protein Kinases-AMPK-Beclin1 in Aerobic Exercise-Induced Autophagy in Middle-Aged Rat Hippocampus

Background: Carbonylation is an oxidative modification of the proteins and a marker of oxidative stress. The accumulation of toxic carbonylated proteins might be one of the onsets of pathogenesis in hippocampal aging or neurodegeneration. Enormous evidence indicates that regular aerobic exercise might alleviate the dysfunction of carbonylated proteins, but the adaptational mechanisms in response to exercise are unclear. Objective: This study explored the carbonyl stress mechanism in the hippocampus using proteomics and the role of calmodulin-dependent protein kinases (CAMK)-AMP-activated protein kinase (AMPK)-Beclin1 signaling pathways in alleviating aging or improving function with regular aerobic exercise. Methods: Twenty-four healthy 13-month-old male Sprague-Dawley rats (average 693.21 ± 68.85 g) were randomly divided into middle-aged sedentary control group (M-SED, n = 12) and middle-aged aerobic exercise runner group (M-EX, n = 12). The M-EX group participated in regular aerobic exercise – treadmill running – with exercise intensity increasing gradually from 50–55% to 65–70% of maximum oxygen consumption (V˙O2max) over 10 weeks. The targeted proteins of oxidative modification were profiled by avidin magnetic beads and electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOF-MS). Western blots were used to test for molecular targets. Results: Regular aerobic exercise restores the intersessional habituation and rescues the hippocampus morphological structure in middle-aged rats. ­ESI-Q-TOF-MS screened 56 carbonylated proteins only found in M-SED and 16 carbonylated proteins only found in M-EX, indicating aerobic exercise decreased carbonyl stress. Intriguingly, Ca²⁺/CAMK II alpha (CAMKIIα) was carbonylated only in the M-SED group at the oxidative modification site of 4-hydroxynonenal adducts, while regular aerobic exercise alleviated CAMKIIα carbonylation. Regular aerobic exercise significantly increased the expression and phosphorylated, active levels of CAMKIIα and AMPKα1. It also upregulated the expression of Beclin1 and microtubule-associated protein1-light chain 3 in the hippocampus. Conclusion: Quantification of CAMKIIα carbonylation may be a potential biomarker of the hippocampal senescence. Additionally, regular aerobic exercise-induced autophagy via the activation of CAMK-AMPK-Beclin1 signaling pathway may mitigate the hippocampal neurodegeneration or pathological changes by alleviating protein carbonylation (carbonyl stress).

Identification and availability of peptides from lactoferrin in the gastrointestinal tract of mice.

Lactoferrin is a protein with multifunctional bioactivities. However, limited studies have reported the digestion behavior of lactoferrin in mice. In the present study, the metabolic fate of lactoferrin in the gastrointestinal tract (GIT) of mice was investigated. The mice were orally administered 4 mg of lactoferrin. The contents in the stomach, duodenum, jejunum and ileum were collected at 0.5 h, 1 h, 2 h and 3 h after administration. The peptides in the contents of the stomach, duodenum, jejunum and ileum were identified by electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOF-MS) coupled with capillary electrophoresis (CE). At 0.5 h after administration, 350, 47, 118 and 26 novel peptides were identified in the contents of the stomach, duodenum, jejunum and ileum, respectively. At 1 h after administration, 175, 23, 3 and 8 novel peptides were identified in the contents of the stomach, duodenum, jejunum and ileum, respectively. At 2 h and 3 h after administration, 96 and 2 novel peptides were identified in the contents of the stomach, respectively. Among these peptides, 10 peptides were generated from gastrointestinal digestions and could play physiological roles when they are absorbed through the intestine in their original form. This work provided some theoretical basis for clarifying the bioactive mechanisms of lactoferrin.

Separation of nine novel triterpene saponins from Camellia japonica seeds using high-performance countercurrent chromatography and reversed-phase high-performance liquid chromatography.

Camellia japonica L. (Theaceae) is an evergreen shrub, which is cultivated as a popular ornamental tree in Korea, China, and Japan and its seeds have been used as a source of cooking oil, in cosmetics and as a traditional medicine. Intensive phytochemical works have revealed that oleanane-type saponins are the characteristic compounds of the seeds of C. japonica. The purpose of the present study is to isolate and determine oleanane-type saponins from C. japonica using high-performance countercurrent chromatography (HPCCC) coupled with reversed-phase high-performance liquid chromatography (RP-HPLC) and spectroscopic evidences, respectively. HPLC electrospray ionisation quadrupole time-of-flight mass spectrometry (ESI-Q-TOF-MS) was applied to profile the saponin composition of an enriched saponin extract of C. japonica seeds. The enriched saponin extract was separated by HPCCC using a dichloromethane/methanol/isopropanol/water (9:6:1:4, v/v/v/v) system and RP-HPLC. The structures of the isolates were determined utilising ESI-Q-TOF-MS, one-dimensional and two-dimensional NMR and optical rotation. HPCCC on enriched saponin extract of C. japonica yielded four saponin fractions in the order of the number of sugars attached to the triterpene aglycone, and preparative RP-HPLC on each saponin fraction led to the isolation of nine novel saponins, namely camoreoside A-I, along with six known ones. This study indicates that combination of HPLC-ESI-Q-TOF-MS analysis and HPCCC coupled with RP-HPLC are excellent tools for discovering saponins from natural sources.

Identification and quantification of product-related quality attributes in bio-therapeutic monoclonal antibody via a simple, and robust cation-exchange HPLC method compatible with direct online detection of UV and native ESI-QTOF-MS analysis

Modern analytical ion-exchange chromatography is one of the conventional tools used for assessment of product-related quality attributes in bio-therapeutic monoclonal antibodies (mAbs). Here, we present an approach to resolve, identify, and quantify product-related substances of therapeutic mAb at its intact molecular level by cation exchange (CIEX) HPLC coupled directly to electrospray ionization - quadrupole time of flight mass spectrometry (ESI-QTOF-MS). This method utilizes pH gradient elution mode comprised of ammonium formate buffer components, and a weak cation exchange column as stationary phase. Furthermore, ion-mobility mass spectrometry (IM-MS) provided additional insights on its higher order structure. Also, orthogonal assays such as conventional CIEX-HPLC, high resolution capillary isoelectric focusing, peptide mapping, spectroscopic, and fluorescence methods were used considerably to support the findings. Additionally, an in vitro assay was included to assess the associated impact on Fc mediated function. Overall, the developed method with simultaneous detection of UV peak area percentage at 280 nm and native ESI-MS is found to be a rapid and robust analytical tool for direct assessment of structural and purity attributes, process optimization, product development, and to decipher the relevant role of micro-variants on quality, stability, and clinical outcomes.

Silver(I) and zinc(II) complexes with symmetrical cinnamaldehyde Schiff base derivative: Spectroscopic, powder diffraction characterization, and antimycobacterial studies

Silver(I) and zinc(II) complexes with N,N′-bis(trans-cinnamaldehyde)ethane-1,2-diamine (EnCinn) have been synthesized and characterized by elemental analysis, simultaneous thermogravimetric and differential thermal studies (TG-DTA), infrared (IR), 1H and 13C nuclear magnetic resonance (NMR) spectroscopic measurements and electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-QTOF-MS). Crystal and molecular structure descriptions were performed based on powder X-ray diffraction data. The AgEnCinn complex has a polymeric structure where the ligand bridges between two Ag(I) ions, while the ZnEnCinn complex has a monomeric structure with EnCinn acting as chelating ligand. Antimycobacterial assays over Mycobacterium tuberculosis showed the activity of the Ag(I) complex with Minimum Inhibitory Concentration (MIC) value of 22.7 μmol L−1, while the Zn(II) complex was shown to be inactive over the same experimental conditions.

Simultaneous qualitative and quantitative evaluation of Ilex kudingcha C. J. tseng by using UPLC and UHPLC-qTOF-MS/MS

In this study, a systematic method was established for the holistic quality control of Ilex kudingcha C. J. Tseng, a popular functional drink for adjuvant treatment of diabetes, hypertension, obesity and hyperlipidemia. Both qualitative and quantitative analyses were conducted. For qualitative analysis, an ultra high performance liquid chromatography (UHPLC) coupled with an electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-qTOF-MS) method was established for rapid separation and structural identification of the constituents in Ilex kudingcha. Samples were separated on an ACQUITY UPLC HSS T3C18 column (2.1 mm × 100 mm, 1.8 μm) by gradient elution using 0.1% (v/v) formic acid (solvent A) and acetonitrile (solvent B) as mobile phases at a flow rate of 0.25 mL min−1. The chromatographic profiling of Ilex kudingcha by UHPLC-qTOF-MS/MS resulted in the characterization of 53 compounds, comprising 18 compounds that were unambiguously identified by comparison with reference standards. For quantitative analysis, 18 major compounds from 15 batches of Ilex kudingcha samples were simultaneously detected by UPLC-DAD at wavelengths of 210 nm, 260 nm, and 326 nm. The method was validated with respect to precision, linearity, repeatability, stability, accuracy, and so on. The contents of the 18 target compounds were applied for hierarchical clustering analysis (HCA) and principal component analysis (PCA) to differentiate between the samples. The results of HCA and PCA were consistent with each other. Sample No. 1 differed significantly based on HCA and PCA, and the differentiating components were confirmed to originate from different batches of samples. Phenolic acids and triterpenes were found to be the main ingredients in Ilex kudingcha. This strategy was effective and straightforward, and provided a potential approach for holistic quality control of Ilex kudingcha.

Arsenic speciation in tree moss by mass spectrometry based hyphenated techniques

A method based on ion-pair reversed phase high performance liquid chromatography (HPLC) hyphenated with inductively coupled plasma mass spectrometry (ICP-MS) was developed for arsenic speciation in extract of tree moss. Under the optimal conditions, the limit of detection of eight arsenic species including arsenite (AsIII), arsenate (AsV), monomethylarsonic acid (MMA), dimethylarsonic acid (DMA), trimethylarsinoxide (TMAO), tetramethylarsonium (Tetra), arsenocholine (AsC) and arsenobetaine (AsB) is between 0.04 and 0.07 ng/mL, with a linear range of 0.2 − 500 ng/mL. Three unknown arsenic species (Unk1, Unk2 and Unk3) and six specific arsenic species (AsIII, AsV, DMA, TMAO, Tetra and AsB) were detected in the extract of tree moss. Unk3 was identified as a kind of arsenosugars (2,3-dihydroxypropyl-5-deoxy-5(dimethylarsenoso)furanoside, arsenosugar X) by electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-qTOF-MS).

Characteristic NH3 and CO losses from sodiated peptides C-terminated by glutamine residues.

Under certain conditions some amino acid (AA) residues undergo special reactions in the gas phase, generating characteristic neutral losses and product ions. Taking these special fragments into account and understanding the effect of AA residues on peptide cleavages will consummate database search algorithms and manual data interpretation in peptide sequencing by mass spectrometry (MS). In this study, the details of the characteristic NH3 and CO losses of glutamine (Gln) residues located at the C-terminus of peptides are presented. A number of selected peptides were fragmented under collision-induced dissociation (CID) in electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-QTOF-MS). Density functional theory (DFT) quantum mechanical calculations at the B3LYP/6-31+G(d,p) level were carried out to optimize the geometry of peptide ions and provide energy barriers of ions in each step during fragmentations. Two characteristic peaks appear near the precursor ions of sodiated Gln C-terminated peptides, suggesting the loss of neutral NH3 and CO via a two-step process. The proposed mechanism of their formation is as follows: after losing NH3 , a non-classical bn * ion is formed with a glutaric anhydride structure that further dissociates to lose CO. The sodiated peptides show more intensive peaks corresponding to the loss of neutral molecules than the protonated ones. This type of neutral loss can also occur at the Gln residue that is rearranged to the C-terminus of sodiated peptides. The experiments and calculations suggest that the two-step characteristic NH3 and CO loss of sodiated peptides is energetically favored, and can be applied to identify C-terminated Gln residues. This study provides a mechanistic insight into the role of sodium ion during peptide fragmentation. Copyright © 2017 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.