A Specific HPLC Method to Determine Residual HEPES in [68Ga]Ga-Radiopharmaceuticals: Development and Validation

Background: Nowadays, in Nuclear Medicine, clinically applied radiopharmaceuticals must meet quality release criteria such as high radiochemical purity and radiochemical yield. Many radiopharmaceuticals do not have marketing authorization and have no dedicated monograph within European Pharmacopeia (Ph. Eur.); therefore, general monographs on quality controls (QCs) have to be applied for clinical application. These criteria require standardization and validation in labeling and preparation, including quality controls measurements, according to well defined standard operation procedures. However, QC measurements are often based on detection techniques that are specific to a certain chromatographic system. Several radiosyntheses of [⁶⁸Ga]Ga-radiopharmaceuticals are more efficient and robust when they are performed with 2-[4-(2-hydroxyethyl)piperazin-1-yl] ethanesulfonic acid (HEPES) buffer, which is considered as an impurity to be assessed in the QC procedure, prior to clinical use. Thus, Ph. Eur. has introduced a thin-layer chromatography (TLC) method to quantify the HEPES amount that is present in [⁶⁸Ga]Ga-radiopharmaceuticals. However, this is only qualitative and has proven to be unreliable. Here we develop and validate a new high-performance liquid chromatography (UV-Radio-HPLC) method to quantify the residual amount of HEPES in ⁶⁸Ga-based radiopharmaceuticals. Method: To validate the proposed UV-Radio-HPLC method, a stepwise approach was used, as defined in the guidance document that was adopted by the European Medicines Agency (CMP/ICH/381/95 2014). The assessed parameters are specificity, linearity, precision (repeatability), accuracy, and limit of quantification. A range of concentrations of HEPES (100, 80, 60, 40, 20, 10, 5, 3 μg/mL) were analyzed. Moreover, to test the validity and pertinence of our new HPLC method, we analyzed samples of [⁶⁸Ga]Ga-DOTATOC; [⁶⁸Ga]Ga-PSMA; [⁶⁸Ga]Ga-DOTATATE; [⁶⁸Ga]Ga-Pentixafor; and [⁶⁸Ga]Ga-NODAGA-Exendin-4 from different batches that were prepared for clinical use. Results: In the assessed samples, HEPES could not be detected by the TLC method that was described in Ph. Eur. within 4 min incubation in an iodine-saturated chamber. Our developed HPLC method showed excellent linearity between 3 and 100 μg/mL for HEPES, with a correlation coefficient (R²) for calibration curves that was equal to 0.999, coefficients of variation (CV%) < 2%, and percent deviation value of bias from 100% to 5%, in accordance with acceptance criteria. The intra-day and inter-day precision of our method was statistically confirmed and the limit-of-quantification (LOQ) was 3 μg/mL, confirming the high sensitivity of the method. The amount of HEPES that was detected with our developed HPLC method in the tested [⁶⁸Ga]Ga-radiopharmaceuticals resulted well below the Ph. Eur. limit, especially for [⁶⁸Ga]Ga-NODAGA-Exendin-4. Conclusions: The TLC method that is described in Ph. Eur. to assess residual HEPES in [⁶⁸Ga]-based radiopharmaceuticals may not be sufficiently sensitive and thus unsuitable for QC release. Our new HPLC method was sensitive, quantitative, reproducible, and rapid for QCs, allowing us to exactly determine the residual HEPES amount in [⁶⁸Ga]Ga-radiopharmaceuticals for safe patient administration.     

Effect of Biodegradable Coatings on the Growth of Aspergillus flavus In Vitro,on Maize Grains,and on the Quality of Tortillas during Storage

The fungus Aspergillus flavus causes serious damage to maize grains and its by-products, such as tortilla. Currently, animal and plant derivatives, such as chitosan and propolis, and plant extract residues, respectively, are employed as alternatives of synthetic fungicides. The objective of this research was to evaluate the efficacy of several formulations based on propolis-chitosan-pine resin extract on the in vitro growth of A. flavus, the growth of maize grain plantlets and the quality of stored tortillas at 4 and 28 °C. The most outstanding formulation was that based on 59.7% chitosan + 20% propolis nanoparticles + 20% pine resin extract nanoparticles; since the in vitro conidia germination of A. flavus did not occur, disease incidence on grains was 25–30% and in tortillas, 0% infection was recorded, along with low aflatoxin production (1.0 ppb). The grain germination and seedling growth were markedly reduced by the nanocoating application. The percentage weight loss and color of tortillas were more affected by this coating compared to the control, and the rollability fell within the scale of non-ruptured at 4 °C and partially ruptured at 28 °C. The next step is to evaluate the toxicity of this formulation.     

A Silver Monochrome “Concetto spaziale” by Lucio Fontana: A Spectroscopic Non- and Micro-Invasive Investigation of Materials

In several of his artworks, for instance the Venezie cycle, Fontana employed metallic paints; previous investigations on such materials highlighted the use of different synthetic binders and of thick paint layers below the metal one, having different colours to change the visual perception of the metallic surface. In the present work, a monochrome silver “Concetto spaziale” by the Italo–Argentine artist belonging to a private collection recently gifted to the museum of the Church of San Fedele in Milano, Italy, was investigated to deepen the knowledge of this particular group of Fontana’s paintings. The artwork was initially visually inspected in visible and ultraviolet (UV) light. Subsequently, a non-invasive spectroscopic investigation was performed by X-ray fluorescence (XRF), reflection Fourier-transform infrared (FTIR) and Raman spectroscopy. A minute fragment of silver-coloured paint was taken from the reverse of the painting, near the cut edge, and examined by scanning electron microscopy coupled with energy dispersive X-ray analysis (SEM-EDX) and micro-Fourier-transform Raman (FT-Raman) spectroscopy. The analytical data made it possible to identify the composition of the metallic paint layer and of the underlying dark one, both from the point of view of the pigments and of the binders used, also highlighting the potential of the non-invasive and micro-invasive methods adopted in the investigation.     

A longitudinal study of MR diffusion changes in normal appearing white matter of patients with early multiple sclerosis

BACKGROUND AND PURPOSE: The stage at which normal appearing white matter (NAWM) abnormalities first appear in multiple sclerosis (MS) is not clear. The aim of our study was to monitor water diffusion changes over time in NAWM of patients with early MS.METHODS: Out of a consecutive series of patients enrolled in a MR study on clinically isolated syndrome (CIS), we selected 19 subjects who had completed a one year follow-up. The MR scans obtained at baseline and at 12 months were reviewed according to the new criteria on the diagnosis of MS. Lesion load on T2 and T1 weighted images and the trace of the apparent diffusion coefficient in NAWM were measured both at baseline and at 12 months in patients and in 12 healthy controls.RESULTS: In three patients the diagnosis of MS was done at baseline based on MR. Thirteen patients developed MS during the study and in three patients the diagnosis remained "possible MS." TADC in NAWM in patients was significantly higher than in controls at the 12 months' follow-up but not at baseline (controls mean tADC +/- sd = 0.745 +/- 0.02 mm(2)/sec x 10(-3); patients mean tADC(12) +/- sd = 0.767 +/- 0.02 mm(2)/sec x 10(-3); p < 0.02). TADC and T2 lesion load at 12 months were significantly correlated (p < 0.01). Patients exhibiting tADC(12) above a confidence interval had a significantly greater EDSS score at the same time period (EDSS(12) +/- sd = 1.9 +/- 0.5 and = 1.1 +/- 0.4 respectively; p < 0.01).CONCLUSIONS: This study suggests that diffusion MR cannot detect alterations in NAWM of patients with a CIS suggestive of MS. After one year, when most patients develop MS, diffusion MR abnormalities in NAWM become apparent. These abnormalities are correlated with T2 lesion load and may contribute to neurological impairment.     

Low n-6/n-3 Gestation and Lactation Diets Influence Early Performance,Muscle and Adipose Polyunsaturated Fatty Acid Content and Deposition,and Relative Abundance of Proteins in Suckling Piglets

Elevated omega-6 (n-6) and omega-3 (n-3) polyunsaturated fatty acids (PUFAs) ratios in swine diets can potentially impose a higher risk of inflammatory and metabolic diseases in swine. A low ratio between the two omega PUFAs has beneficial effects on sows’ and piglets’ production performance and immunity status. At present, there are few studies on how sow nutrition directly affects the protein and fat deposition in suckling piglets. Two groups of sows were fed diets with high or low n-6/n-3 polyunsaturated ratios of 13:1 (SOY) and 4:1 (LIN), respectively, during gestation and lactation. Longissimus dorsi muscle and adipose tissue from newborn piglets, nourished only with sow’s milk, were subjected to fatty acid profiling by gas chromatography–mass spectrometry (GC-MS) and to proteomics assays based on nano-liquid chromatography coupled to high-resolution tandem mass spectrometry (nLC-HRMS). Fatty acid profiles on both muscle and adipose tissues resembled the magnitude of the differences between fatty acid across diets. Proteomic analysis revealed overabundance of 4 muscle and 11 adipose tissue proteins in SOY compared to LIN in both piglet tissues. The detected overabundance of haptoglobin, an acute-phase protein, and the stimulation of protein-coding genes and proteins related to the innate immune response and acute inflammatory response could be associated with the pro-inflammatory role of n-6 PUFAs.     

Combined prostate diffusion tensor imaging and dynamic contrast enhanced MRI at 3T — quantitative correlation with biopsy

The purpose of this work was to compare diagnostic accuracy of Diffusion Tensor Imaging (DTI), dynamic contrast-enhanced magnetic resonance imaging (DCE MRI) and their combination in diagnosing prostate cancer. Twenty-five patients with clinical suspicion of prostate cancer underwent MRI, prior to transrectal ultrasound-guided biopsies. MRI data were correlated to biopsy results. Logistic regression models were constructed for the DTI parameters, DCE MRI parameters, and their combination. The areas under the receiver operator characteristic curves (AUC) were compared between the models. The nonparametric Wilcoxon signed rank test was used for statistical analysis. The sensitivity and specificity values were respectively 81% (74–87%) and 85% (79–90%) for DTI and 63% (55–70%) and 90% (85–94%) for DCE. The combination “DTI or DCE MRI” had 100% (97–100%) sensitivity and 77% (69–83%) specificity, while “DTI and DCE MRI” had 44% (37–52%) sensitivity and 98% (94–100%) specificity. The AUC for DTI+DCE parameters was significantly higher than that for either DTI (0.96 vs. 0.92, P=.0143) or DCE MRI parameters (0.96 vs. 0.87, P=.00187) alone. In conclusion, the combination of DTI and DCE MRI has significantly better accuracy in prostate cancer diagnosis than either technique alone.     

Characterization of nanochannel delivery membrane systems for the sustained release of resveratrol and atorvastatin: new perspectives on promoting heart health

Novel drug delivery systems capable of continuous sustained release of therapeutics have been studied extensively for use in the prevention and management of chronic diseases. The use of these systems holds promise as a means to achieve higher patient compliance while improving therapeutic index and reducing systemic toxicity. In this work, an implantable nanochannel drug delivery system (nDS) is characterized and evaluated for the long-term sustained release of atorvastatin (ATS) and trans-resveratrol (t-RES), compounds with a proven role in managing atherogenic dyslipidemia and promoting cardioprotection. The primary mediators of drug release in the nDS are nanofluidic membranes with hundreds of thousands of nanochannels (up to 100,000/mm²) that attain zero-order release kinetics by exploiting nanoconfinement and molecule-to-surface interactions that dominate diffusive transport at the nanoscale. These membranes were characterized using gas flow analysis, acetone diffusion, and scanning and transmission electron microscopy (SEM, TEM). The surface properties of the dielectric materials lining the nanochannels, SiO₂ and low-stress silicon nitride, were further investigated using surface charge analysis. Continuous, sustained in vitro release for both ATS and t-RES was established for durations exceeding 1 month. Finally, the influence of the membranes on cell viability was assessed using human microvascular endothelial cells. Morphology changes and adhesion to the surface were analyzed using SEM, while an MTT proliferation assay was used to determine the cell viability. The nanochannel delivery approach, here demonstrated in vitro, not only possesses all requirements for large-scale high-yield industrial fabrication, but also presents the key components for a rapid clinical translation as an implantable delivery system for the sustained administration of cardioprotectants.     

On the determination of the point of fracture initiation by the load separation criterion in J-testing of ductile polymers

The application of an experimental approach based on the load separation criterion for the determination of the point of fracture initiation in a fracture test on a ductile polymer was critically examined. To this aim, the fracture process outlined by the application of this method was related to that described by the visual analysis of the fracture surfaces obtained in fracture tests on nominally identical specimens, in which different levels of crack extension were produced. The material examined was an acrylonitrile-butadiene-styrene (ABS) resin, and the fracture tests were performed at low loading rate on single-edge notched in bending specimens.The results demonstrated that this load separation criterion based methodology is a promising approach for the determination of the point of fracture initiation, and for material fracture resistance, J_Ic, evaluation. The method also has experimental simplicity and a high degree of repeatability.     

Trace elements in sediments and bioaccumulation in European silver eels (Anguilla anguilla L.) from a Mediterranean lagoon (SE Italy)

Samples of surface sediments and tissues (liver and muscle) of commercially available European silver eels (Anguilla anguilla L.) collected from Varano lagoon (Italy) were analysed to determine trace element contents. Univariate and multivariate analyses were performed to highlight both the differences between sampling sites and the influence of channel discharges. Atomic ratios indices for sediment data and biological enrichment factors (BEF) for eel tissues were calculated in order to evaluate the enrichment factor due to human activities. The highest levels of As (11.9?µg?g^?1) and Zn (14.1?µg?g^?1) were observed in the south-eastern zone of the lagoon, which is influenced by urban and agricultural discharges. The low levels of Hg observed in this study (0.04?µg?g^?1) led us to exclude both natural and human local sources of this element. Trace element concentrations of all elements were lower in muscle than in liver tissue. Significant enrichment of Cu and Zn was found in livers.     

A general synthesis of diversely substituted indazoles and hetero-aromatic derivatives from o-halo-(het)arylaldehydes or -phenones

A set of variously substituted indazoles and hetero-aromatic derivatives were synthesized from o-halo-(het)arylaldehydes using a palladium catalyzed amination followed by cyclization. Starting from phenones, this process was extended to give 3-substituted indazoles. Moreover, N-1-substituted-indazoles can be reached by this strategy using an optional selective N-1-alkylation step during the process. This methodology offers a general and easy route for the synthesis of regioselectively substituted indazoles.