Study of immobilized metal affinity chromatography sorbents for the analysis of peptides by on‐line solid‐phase extraction capillary electrophoresis‐mass spectrometry

Several commercial immobilized metal affinity chromatography sorbents were evaluated in this study for the analysis of two small peptide fragments of the amyloid β‐protein (Aβ) (Aβ(1–15) and Aβ(10–20) peptides) by on‐line immobilized metal affinity SPE‐CE (IMA‐SPE‐CE). The performance of a nickel metal ion (Ni(II)) sorbent based on nitrilotriacetic acid as a chelating agent was significantly better than two copper metal ion (Cu(II)) sorbents based on iminodiacetic acid. A BGE of 25 mM phosphate (pH 7.4) and an eluent of 50 mM imidazole (in BGE) yielded a 25‐fold and 5‐fold decrease in the LODs by IMA‐SPE‐CE‐UV for Aβ(1–15) and Aβ(10–20) peptides (0.1 and 0.5 μg/mL, respectively) with regard to CE‐UV (2.5 μg/mL for both peptides). The phosphate BGE was also used in IMA‐SPE‐CE‐MS, but the eluent needed to be substituted by a 0.5% HAc v/v solution. Under optimum preconcentration and detection conditions, reproducibility of peak areas and migration times was acceptable (23.2 and 12.0%RSD, respectively). The method was more sensitive for Aβ(10–20) peptide, which could be detected until 0.25 μg/mL. Linearity for Aβ(10–20) peptide was good in a narrow concentration range (0.25–2.5 μg/mL, R² = 0.93). Lastly, the potential of the optimized Ni(II)‐IMA‐SPE‐CE‐MS method for the analysis of amyloid peptides in biological fluids was evaluated by analyzing spiked plasma and serum samples.     

Exosome enrichment of human serum using multiple cycles of centrifugation

In this work, we compared the use of repeated cycles of centrifugation at conventional speeds for enrichment of exosomes from human serum compared to the use of ultracentrifugation (UC). After removal of cells and cell debris, a speed of 110 000 × g or 40 000 × g was used for the UC or centrifugation enrichment process, respectively. The enriched exosomes were analyzed using the bicinchoninic acid assay, 1D gel separation, transmission electron microscopy, Western blotting, and high‐resolution LC‐MS/MS analysis. It was found that a five‐cycle repetition of UC or centrifugation is necessary for successful removal of nonexosomal proteins in the enrichment of exosomes from human serum. More significantly, 5× centrifugation enrichment was found to provide similar or better performance than 5× UC enrichment in terms of enriched exosome protein amount, Western blot band intensity for detection of CD‐63, and numbers of identified exosome‐related proteins and cluster of differentiation (CD) proteins. A total of 478 proteins were identified in the LC‐MS/MS analyses of exosome proteins obtained from 5× UCs and 5× centrifugations including many important CD membrane proteins. The presence of previously reported exosome‐related proteins including key exosome protein markers demonstrates the utility of this method for analysis of proteins in human serum.     

Crystallization and melting behaviour of poly(m-xylene adipamide)

The melting and crystallisation behaviour of poly(m-xylene adipamide) (MXD6) are investigated by using the conventional DSC, X-ray diffraction and polarised light microscopy. Triple, double or single melting endotherms are obtained in subsequent heating scan for the samples after isothermal crystallisation from the melt state at different temperatures. The lowest melting peak can be ascribed to the melting of secondary crystals. The melting of primary crystals causes the medium melting peak and the highest melting peak is attributed to the melting of recrystallised species formed during heating. Following the Hoffman–Weeks theory, the equilibrium melting temperature is equal to 250°C and the equilibrium melting enthalpy ΔH _m ⁰ to 175 J g^–1. Then, using the Lauritzen–Hoffmann theory of secondary crystallisation, the analyse of the spherulitic growth shows that the temperature of transition between the growing regimes II and III is equal to 176°C. Finally the Gibbs-Thomson relationship allows the determination of the distribution function of crystalline lamellae.     

New interface plate for microspray ionization mass spectrometry

A new interface plate was employed in microspray ionization mass spectrometry (μESI-MS) to improve ion transmission from the sprayer into the sampling nozzle of the mass spectrometer at atmospheric pressure. Using a time-of-flight mass spectrometer (TOFMS), a fivefold increase in ion intensity and a sevenfold reduction in method detection limit were observed. The interface plate attenuated the dependence of the ion intensity on the sprayer position. Even when the distance between the sprayer tip and sampling nozzle was 15.0 mm, ion signals were still stronger than when the sprayer tip was positioned 3.0 mm in front of the sampling nozzle with the original interface plate. This enhancement in the performance of μESI-MS was due to the improved shapes of the equipotential lines near the sprayer tip and the long desolvation distance between the sprayer and the sampling nozzle of the MS.     

Silicon Analogues of Triarylmethanol Hosts. Inclusion Properties and Host–guest Structures: A Comparative Study

The simple triarylmethanol hosts, 2 and 4, and their silicon analogues, 1 and 3, have been studied for comparison of the formation of crystalline inclusion compounds. Clathrate formation experiments showed that replacement of the carbinol C atoms in 2 and 4 by Si atoms to give 1 and 3 resulted in a distinct increase of the capability to form inclusion compounds with organic guests, in particular with alcohols. Moreover, the naphthyl derivatives are much more efficient than the phenyl species, irrespective of the carbinol and silanol features. In order to investigate and compare the guest recognition modes and packing relations of hosts 1–4 in their crystalline inclusion compounds, 11 selected co-crystals, namely 1·DMSO (2:1), 3·EtOH (1:1), 3· i-PrOH (1:1), 3·acetone (1:1), 3·DMSO (1:1), 3·THF (1:1), 3·piperidine (1:1), 4·acetone (1:1), 4·DMSO (1:1), 4·1,4-dioxane (1:1) and 4·benzene (1:1), were studied by X-ray diffraction from single crystals. The survey contains additional 11 crystal structures from the literature and provides a detailed discussion of isostructurality relationships.Supplementary Data relevant to this publication have been deposited with the Cambridge Crystallographic Data Centre as supplementary publications nos. CCDC 274780–274790.     

Determination of compound-specific Hg isotope ratios from transient signals using gas chromatography coupled to multicollector inductively coupled plasma mass spectrometry (MC-ICP/MS)

MeHg and inorganic Hg compounds were measured in aqueous media for isotope ratio analysis using aqueous phase derivatization, followed by purge-and-trap preconcentration. Compound-specific isotope ratio measurements were performed by gas chromatography interfaced to MC-ICP/MS. Several methods of calculating isotope ratios were evaluated for their precision and accuracy and compared with conventional continuous flow cold vapor measurements. An apparent fractionation of Hg isotopes was observed during the GC elution process for all isotope pairs, which necessitated integration of signals prior to the isotope ratio calculation. A newly developed average peak ratio method yielded the most accurate isotope ratio in relation to values obtained by a continuous flow technique and the best reproducibility. Compound-specific isotope ratios obtained after GC separation were statistically not different from ratios measured by continuous flow cold vapor measurements. Typical external uncertainties were 0.16‰ RSD (n = 8) for the ²⁰²Hg^/198Hg ratio of MeHg and 0.18‰ RSD for the same ratio in inorganic Hg using the optimized operating conditions. Using a newly developed reference standard addition method, the isotopic composition of inorganic Hg and MeHg synthesized from this inorganic Hg was measured in the same run, obtaining a value of δ ²⁰²Hg = −1.49 ± 0.47 (2SD; n = 10). For optimum performance a minimum mass of 2 ng per Hg species should be introduced onto the column.     

Crystal structure, phase transition and thermal behaviour of dabcodiium hexaaquacopper(II) bis(sulfate), (C6H14N2)[Cu(H2O)6](SO4)2

A new organically templated metal sulfate has been synthesized and characterized. At room temperature, dabcodiium hexaaquacopper(II) bis(sulfate), (C₆H₁₄N₂)[Cu(H₂O)₆](SO₄)₂ crystallizes in the monoclinic symmetry (space group P2₁/n) with the following unit cell parameters: a = 6.9533(2), b = 12.5568(2), c = 9.9434(2) Å; β = 90.526(1)° and Z = 2. Its crystal structure is built from isolated [Cu(H₂O)₆]²⁺, and disordered ions linked together by a hydrogen-bonding network. The title compound undergoes a reversible phase transition of the first-order type at 265.7/281.8 K on heating–cooling runs. Below the phase transition temperature, the structure is fully ordered.     

Electrochemical characterization of glassy carbon electrodes modified with hybrid inorganic-organic single-layer of α-Keggin type polyoxotungstates

It has been demonstrated, for the first time, that an adsorbed single-layer of the hybrid salts (TBA)₄H₃PW₁₁O₃₉, (TBA)₄PW₁₁Fe(H₂O)O₃₉, (TBA)₄PW₁₁Mn(H₂O)O₃₉ and (TBA)₄HPW₁₁Co(H₂O)O₃₉ can be fabricated on the surface of a glassy carbon electrode by the droplet evaporation methodology. These chemically modified electrodes were stable and their preparation was reproducible and easy to perform. The electrochemical features of the immobilized polyanions were different from those of the corresponding soluble species, namely in what concerns the peak potential values. The effect of the scan rate and of pH on the voltammetric features led to the conclusion that the first W reduction process for all immobilized polyanions was diffusion-controlled. For TBA-PW₁₁, TBA-PW₁₁Fe and TBA-PW₁₁Co the two-electron reductions at the first W waves are accompanied by the uptake of protons (2 H⁺ for the PW₁₁ anion and 4 H⁺ for the Fe-substituted and Co-substituted species). For the PW₁₁Mn-modified electrode, the reduction at the first W wave was a 1 e/2 H⁺ process. Additionally, the results obtained in the presence of Na₂SO₄ in the solution highlighted the role of the ions in the supporting electrolyte in the redox features of the immobilized hybrid phosphotungstates.     

Untargeted fatty acid profiles based on the selected ion monitoring mode

Fatty acids are potential biomarkers of some diseases and also key markers and quality parameters of different dietary fats and related products. Thus, untargeted fatty acid profiles are important in the study of dietary fat quality and fat-related diseases, as well as in other fields such as bioenergy. In addition, accurate identification of unknown components is a technological breakthrough for the selected ion monitoring (SIM) mode for untargeted profiles. In this study, we developed untargeted fatty acid profiles based on SIM. We also investigated mass spectral characteristics and equivalent chain lengths (ECL) to eliminate the influence of non-FAMEs for identifying fatty acids in samples. As an application example, fatty acid profiles were used to classify three edible vegetable oils. The results indicated that SIM-based untargeted fatty acid profiles could yield accurate qualitative and quantitative results for more fatty acids and benefit related studies of metabolite profiles.     

2-(2-Phenyl-1H-phenanthro-[9,10-d]imidazole-1-yl)-acetic acid (PPIA) and its application for determination of amines by high performance liquid chromatography with fluorescence detection and identification with mass spectroscopy/atmospheric pressure chemical ionization

A simple, sensitive, and mild method for the determination of amino compounds based on a condensation reaction with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC·HCl) as the dehydrant with fluorescence detection has been developed. Amines were derivatized to their acidamides with labeling reagent 2-(2-phenyl-1H-phenanthro-[9,10-d]imidazole-1-yl)-acetic acid (PPIA). Studies on derivatization conditions indicated that the coupling reaction proceeded rapidly and smoothly in the presence of a base catalyst in acetonitrile to give the corresponding sensitively fluorescent derivatives with an excitation maximum at λ_ex 260 nm and an emission maximum at λ_em 380 nm. The labeled derivatives exhibited high stability and were enough to be efficiently analyzed by high-performance liquid chromatography. Identification of derivatives was carried out by online post-column mass spectrometry (LC/APCI-MS/MS) and showed an intense protonated molecular ion corresponding m/z [MH]⁺ under APCI in positive-ion mode. At the same time, the fluorescence properties of derivatives in various solvents or at different temperature were investigated. The method, in conjunction with a gradient elution, offered a baseline resolution of the common amine derivatives on a reversed-phase Eclipse XDB-C₈ column. LC separation for the derivatized amines showed good reproducibility with acetonitrile-water as mobile phase. Detection limits calculated from 0.78 pmol injection, at a signal-to-noise ratio of 3, were 3.1-18.2 fmol. The mean intra- and inter-assay precision for all amine levels were <3.85% and 2.11%, respectively. Excellent linear responses were observed with coefficients of >0.9996. The established method for the determination of aliphatic amines from real wastewater and biological samples was satisfactory.