Quantitation of a PEGylated protein in monkey serum by UHPLC-HRMS using a surrogate disulfide-containing peptide: A new approach to bioanalysis and in vivo stability evaluation of disulfide-rich protein therapeutics

To quantify a therapeutic PEGylated protein in monkey serum as well as to monitor its potential in vivo instability and methionine oxidation, a novel ultra high performance liquid chromatography-high resolution mass spectrometric (UHPLC-HRMS) assay was developed using a surrogate disulfide-containing peptide, DCP(SS), and a confirmatory peptide, CP, a disulfide-free peptide. DCP(SS) was obtained by eliminating the step of reduction/alkylation before trypsin digestion. It contains an intact disulfide linkage between two peptide sequences that are essential for drug function but susceptible to potential in vivo cleavages. HRMS-based single ion monitoring (SIM) on a Q Exactive™ mass spectrometer was employed to improve assay specificity and sensitivity for DCP(SS) due to its poor fragmentation and low sensitivity with SRM detection. The assay has been validated for the protein drug in monkey serum using both surrogate peptides with excellent accuracy (within ±4.4%Dev) and precision (within 7.5%CV) with a lower limit of quantitation (LLOQ) at 10 ng mL⁻¹. The protein concentrations in monkey serum obtained from the DCP(SS)-based assay not only provided important pharmacokinetic parameters, but also confirmed in vivo stability of the peptide regions of interest by comparing drug concentrations with those obtained from the CP-based assay or from a ligand-binding assay (LBA). Furthermore, UHPLC-HRMS allowed simultaneous monitoring of the oxidized forms of both surrogate peptides to evaluate potential ex vivo/in vivo oxidation of one methionine present in each of both surrogate peptides. To the best of our knowledge, this is the first report of using a surrogate disulfide-containing peptide for LC-MS bioanalysis of a therapeutic protein.     

Existence results for globally efficient solutions of vector equilibrium problems via a generalized kkm principle

The aim of this article is to present new existence results for globally efficient solutions of a strong vector equilibrium problem given by a sum of two functions via a generalized KKM principle, and to establish the connectedness of the solutions set.     

Organo-silica hybrid capillary monolithic column with mesoporous silica particles for separation of small aromatic molecules

Monolithic stationary phases for use in capillary electrochromatography were prepared by incorporation of mesoporous silica particles (of type MCM-41 or UVM-7) in a polymer obtained from butyl methacrylate and ethylene glycol dimethacrylate as monomers, 1,4-butanediol and 1-propanol as porogen, and azobisisobutyronitrile as initiator. The stability of the dispersions with varying fractions of silica particles was investigated by UV-vis spectrometry. Using continuous stirring during the capillary filling and short UV-polymerization times, polymeric beds with homogenously dispersed mesoporous particles (with contents up to 35 wt% of silica) are obtained. The resulting hybrid monolithic columns were characterized using scanning electron microscopy. The chromatographic performance of these novel stationary phases was evaluated by using alkyl benzenes and benzoic acid derivatives as test analytes. The use of these polymers leads to increased retention and separation efficiency compared to the parent monolith. The column efficiency reached values of up to 140,000 plates m^?1. The resulting hybrid monolithic columns also exhibited a satisfactory reproducibility with relative standard deviations of ca. 14% (batch-to-batch).

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Graphical abstract Hybrid polymer monoliths containing large amounts of mesoporous silica-particles (MCM-41 or UVM-7) were prepared by UV initiation. The prepared monolithic columns showed higher retention times and efficiencies than parent monoliths for alkyl benzenes and benzoic acid derivatives.

     

Effects of Redox Couple on the Response of Polypyrrole‐Based Electrochemical DNA Sensors

Much research‐to‐date exists to create sensitive and selective DNA sensors. A variety of approaches are adopted, including those involving polypyrrole and electrical impedance spectroscopy. Combined these are able to sense as well as aptly transduce signals. Many such sensors employ ferri/ferrocyanide to exhibit surface hybridization. This redox reporter, though with merits, can prove disadvantageous. To this end, a comparative study was performed with the redox reporter hydroquinone. For comparison purposes ferri/ferrocyanide indicated greater sensitivity, suggested to arise from inherent charge. Conversely, with hydroquinone steric hindrance is the main influencing factor. Such a study aims to guide design of analogous sensors.     

Comparative analysis of lipophilic compounds in eggs of organically raised ISA Brown and Araucana hens

Hens?? eggs represent a rich source of important nutrients, including lipids and carotenoids. The lipid composition of hens?? eggs is influenced by genetic factors, age, and diet. The aim of this study was to compare the fatty acids, cholesterol, and carotenoids content of the egg yolk of ISA Brown and Araucana hens grown in free-range housing systems. Fatty acids and cholesterol were analysed by GC-FID and GC-MS and carotenoids were quantified by RP-HPLC-PDA. The Araucana egg yolk has a higher lipid content and higher egg-to-albumen ratio than the ISA Brown yolk, while the total cholesterol, carotenoids content and profile are not significantly different. The lipids of the Araucana egg yolk have a higher content of mono-unsaturated fatty acids (MUFAs), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) and a better n-6/n-3 ratio than the ISA Brown egg yolk lipids. The major carotenoids were lutein and zeaxanthin, which account for more than 83 % in egg yolk. Eggs of both breeds, when raised organically, represent very good sources of highly bio-available lutein and zeaxanthin, pigments which are related to lower risk of age-related macular degeneration. We report for the first time on the fatty acids composition in lipid fractions and the profile and content of carotenoids of the Araucana egg yolk.     

Surface‐enhanced Raman spectroscopy of DNA from leaves of in vitro grown apple plants

Ultrasensitive Raman measurements of nucleic acids are possible by exploiting the effect of surface‐enhanced Raman scattering (SERS). In this work, the vibrational spectra of eight genomic DNAs from in vitro grown apple leaf tissues (Malus domestica Borkh., Fam Rosaceae, cvs. Florina, Idared, Rebra, Goldrush, Romus 3, Romus 4 and the rootstocks M9 and M26) were analyzed using surface‐enhanced Raman spectroscopy, in the wavenumber range 200–1800 cm⁻¹. SERS signatures, spectroscopic band assignments and structural interpretations of these plant genomic DNAs are reported. Strong dependences of the SERS spectra on genomic DNA amount in the measured sample volume and on time were observed. Similarities of the SERS signals of DNAs from Rebra and Romus 3 leaves were detected. To our knowledge, this is the first SERS study on genomic DNA from leaf tissues. The present work provides a basis for future use of surface‐enhanced Raman spectroscopy to analyze specific plant DNA–ligand interactions or DNA structural changes induced by plants' stress conditions associated with their natural environment. Besides, this study will generate information that is valuable in the development of low‐level plant DNA‐based analytical sensors. Copyright © 2010 John Wiley & Sons, Ltd.     

The determination of sulfonamides in honey by high performance liquid chromatography--mass spectrometry method (LC/MS)

The sulfonamides (SAs) are stable chemotherapeutics used against the bacterial disease affecting bees, known as American foulbrood (Bacillus larvae), so their residues could appear in the honey of treated bees. Their presence at a concentration above the limit value could be a potential danger to human health. Therefore, a simple, rapid, and reliable method for determination of 11 available SAs in honey was optimized. The samples were homogenized and cleaned with extraction on solid phase by means of Chromabond C18 end-capped cartridge followed by LC/MS analyses. A detection limit of 25 microg/kg was achieved for all analytes. The repeatability of the method was proven and the optimal parameters for temperature and pH of the mobile phase and acetic buffer, respectively, were determined. In this study, 20 samples of domestic honey were included. Six of the analyzed samples were positive, but all results were below the Croatian permissible limit value (100 microg/kg).     

Photoredox Properties of Iron(III) Fluoro Complexes Containing Tetradentate Open-Chain N2O2Ligands

Tetradentate open-chain Schiff base N₂O₂-ligands of acacen, benacen or salen type and fluoride anions F^? coordinate to the iron(III) central atom in methanol forming the complexes [Fe(N₂O₂)(CH₃OH)F]. The complexes do not undergo spontaneous redox changes when kept in the dark. Their irradiation into intraligand or ligand-to-metal charge transfer bands causes the photoreduction of Fe(III) to Fe(II) associated with oxidation of metanol to its radical CH₂OH. The final products of the primary photoredox and secondary dark redox processes, Fe(II) and CH₂O, are formed in a 2:1 molar ratio. The efficiency of the axial methanol ligand photooxidation is strongly wavelength dependent and influenced by the peripheral groups R of the tetradentate ligands