Sequence of polymers by mass spectrometry

Mass Spectrometry, being able to look at the mass of individual molecules in a mixture of homologues, is particularly suitable for the detection of a series of oligomers. However, mass spectra had not been exploited to estimate oligomers distributions, due to the diffuse notion that a lack of correlation existed between peak intensities and concentration of the oligomers in the mixture. The introduction of soft-ionization techniques has largely eliminated this problem. A novel method for the determination of the microstructure of copolymers is presented here. We have recently found that opportune decoding of the information contained in the mass spectral intensities leads to the determination of composition and microstructure in copolymers, and this represents a significant progress. Statistical modeling of the mass spectral intensities of copolymers has been used to derive information on the distribution of monomers along the copolymer chain, and an automated procedure to find the composition and the sequence of the copolymers analyzed has been developed. The statistical analysis of copolymers makes use of Bernoullian and Markovian models in order to characterize the microstructure of copolymer samples, and assuming a theoretical distribution and then fitting the calculated oligomer abundances with the experimental MS peak intensities, the copolymer composition can be determined. A method is also reported to obtain the copolymer conposition by direct analysis of the mass spectra. These theories have been applied to determine the composition and the microstructure of several copolymers whose mass spectra have been reported in the most recent literature.     

The Ferrocenylethynyl Unit: a Stable Hormone Tag

We prepared the organometallic complex 17α‐(ferrocenylethynyl)estradiol (=[(3,17β‐dihydroxyestra‐1,3,5(10)‐trien‐17α‐yl)ethynyl]ferrocene; FcEE; 1 ) by a novel synthetic method. This metallocene possesses sufficient stability in aqueous media to permit the study of its biological properties. Thus, we were able to show that, despite the addition of a bulky substituent at the 17α position of the steroid, the metallocene is still well‐ recognized by an antibody specific to estradiol (CR=40%) and by both subtypes (ERα, ERβ) of the estrogen receptor (at 0°, RBA=28 and 37%, resp.). A DCI‐MS study of the stability of the carbocation [FcEE−OH]⁺ showed moderate stabilization of the carbocation, in agreement with the pK value of −0.72 found for the metallocene by means of Deno's method. The presence of the ferrocene allows the electrochemical detection of FcEE ( 1 ) by HPLC‐ED, with a detection limit of ca. 1 nM , suitable for quantitative pharmacological analysis.     

Interfacial stress analysis and prediction of debonding for a thin plate bonded to a curved substrate

This paper focuses on the analytical and numerical modeling of the interface between a rigid substrate with simple constant curvature and a thin bonded plate. The interfacial behavior is modeled by independent cohesive laws in the normal and tangential directions, coupled with a mixed-mode fracture criterion. The newly developed analytical model determines the interfacial shear and normal stress distributions as functions of the substrate curvature, during the various behavioral stages of the interface prior to the initiation of debonding. The model is also able to predict the debonding load and the effective bond length. In the numerical model the interface is modeled by zero-thickness node-to-segment contact elements, in which both the geometrical relationships between the nodes of the discretized problem and the interface constitutive laws are suitably defined. Numerical results and comparisons between the predictions of the two models are presented.     

Formation of Symmetric Structures in the Dynamics of Repelling Particles

We consider Hamiltonian systems corresponding to the motions of a system of N repelling particles evolving in space under the action deriving from a very long range potential energy; the asymptotic behavior of the system is analysed for the cases U=− ln r and . Only special “asymptotic?shapes” are reached, which may present quite interesting symmetries and correspond to the critical points of a gradient system. The relationships between the original Hamiltonian and the asymptotic gradient system are discussed. Accepted: May 25, 1999     

Behaviour of 5,6-dihydrothieno[2,3-h]cinnolin-3(2H)-one and 5,6-dihydrothieno[3,2-h]cinnolin-3(2H)-one towards hydrazine. Synthesis of thienocinnolinones and of 4-aminothienocinnolinones

The isomeric compounds 5,6-dihydrothieno[2,3-h]cinnolin-3(2H)-one ( 7a ) and 5,6-dihydrothieno-[3,2-h]cinnolin-3(2H)-one ( 7b ) rapidly tautomerise to the corresponding 1,4-dihydrothienocinnolinones 8a,b when kept in refluxing hydrazine hydrate. With longer reaction times the initially formed 8a,b dehydrogenate to the thienocinnolinones 9a,b which eventually are aminated to 4-aminothienocinnolinones 10a,b . This behaviour recalls that reported for the related 5,6-dihydrobenzocinnolin-3(2H)-one ( 1 ) which under the same conditions undergoes dehydrogenation to benzo[h]cinnolin-3(2H)-one ( 2 ) followed by 4-amination to 3 , but differs for the stability of the intermediates, for the mechanism of the final amination, and for the higher reaction rate. All these differences can be rationalised in terms of the heats of formation of the intermediates and products of the two series of transformations.     

Development and Validation of an Analytical HPLC Method to Assess Chemical and Radiochemical Purity of [68Ga]Ga-NODAGA-Exendin-4 Produced by a Fully Automated Method

Background: Glucagon-like peptide 1 receptor (GLP-1R) is preferentially expressed in pancreatic islets, especially in β-cells, and highly expressed in human insulinomas and gastrinomas. In recent years several GLP-1R–avid radioligands have been developed to image insulin-secreting tumors or to provide a tentative quantitative in vivo biomarker of pancreatic β-cell mass. Exendin-4, a 39-amino acid peptide with high binding affinity to GLP-1R, has been labeled with Ga-68 for imaging with positron emission tomography (PET). Preparation conditions may influence the quality and in vivo behavior of tracers. Starting from a published synthesis and quality controls (QCs) procedure, we have developed and validated a new rapid and simple UV-Radio-HPLC method to test the chemical and radiochemical purity of [⁶⁸Ga]Ga-NODAGA-exendin-4, to be used in the clinical routine. Methods: Ga-68 was obtained from a ⁶⁸Ge/⁶⁸Ga Generator (GalliaPharma^®) and purified using a cationic-exchange cartridge on an automated synthesis module (Scintomics GRP^®). NODAGA-exendin-4 contained in the reactor (10 µg) was reconstituted with HEPES and ascorbic acid. The reaction mixture was incubated at 100 °C. The product was purified through HLB cartridge, diluted, and sterilized. To validate the proposed UV-Radio-HPLC method, a stepwise approach was used, as defined in the guidance document released by the International Conference on Harmonization of Technical Requirements of Pharmaceuticals for Human Use (ICH), adopted by the European Medicines Agency (CMP/ICH/381/95 2014). The assessed parameters are specificity, linearity, precision (repeatability), accuracy, and limit of quantification. Therefore, a range of concentrations of Ga-NODAGA-exendin-4, NODAGA-exendin-4 (5, 4, 3.125, 1.25, 1, and 0.75 μg/mL) and [⁶⁸Ga]Ga-NODAGA-exendin-4 were analyzed. To validate the entire production process, three consecutive batches of [⁶⁸Ga]Ga-NODAGA-exendin-4 were tested. Results: Excellent linearity was found between 5–0.75 μg/mL for both the analytes (NODAGA-exendin-4 and ⁶⁸Ga-NODAGA-exendin-4), with a correlation coefficient (R²) for calibration curves equal to 0.999, average coefficients of variation (CV%) < 2% (0.45% and 0.39%) and average per cent deviation value of bias from 100%, of 0.06% and 0.04%, respectively. The calibration curve for the determination of [⁶⁸Ga]Ga-NODAGA-exendin-4 was linear with a R² of 0.993 and CV% < 2% (1.97%), in accordance to acceptance criteria. The intra-day and inter-day precision of our method was statistically confirmed using 10 μg of peptide. The mean radiochemical yield was 45 ± 2.4% in all the three validation batches of [⁶⁸Ga]Ga-NODAGA-exendin-4. The radiochemical purity of [⁶⁸Ga]Ga-NODAGA-exendin-4 was >95% (97.05%, 95.75% and 96.15%) in all the three batches. Conclusions: The developed UV-Radio-HPLC method to assess the radiochemical and chemical purity of [⁶⁸Ga]Ga-NODAGA-exendin-4 is rapid, accurate and reproducible like its fully automated production. It allows the routine use of this PET tracer as a diagnostic tool for PET imaging of GLP-1R expression in vivo, ensuring patient safety.