Synthesis and Biological Evaluation of Flavonoid-Cinnamic Acid Amide Hybrids with Distinct Activity against Neurodegeneration in Vitro and in Vivo.

Flavonoids are polyphenolic natural products and have shown significant potential as disease-modifying agents against neurodegenerative disorders like Alzheimer's disease (AD), with activities even in vivo. Hybridization of the natural products taxifolin and silibinin with cinnamic acid led to an overadditive effect of these compounds in several phenotypic screening assays related to neurodegeneration and AD. Therefore, we have exchanged the flavonoid part of the hybrids with different flavonoids, which show higher efficacy than taxifolin or silibinin, to improve the activity of the respective hybrids. Chemical connection between the flavonoid and cinnamic acid was realized by an amide instead of a labile ester bond to improve stability towards hydrolysis. To investigate the influence of a double bond at the C-ring of the flavonoid, the dehydro analogues of the respective hybrids were also synthesized. All compounds obtained show neuroprotection against oxytosis, ferroptosis and ATP-depletion, respectively, in the murine hippocampal cell line HT22. Interestingly, the taxifolin and the quercetin derivatives are the most active compounds, whereby the quercetin derivate shows even more pronounced activity than the taxifolin one in all assays applied. As aimed for, no hydrolysis product was found in cellular uptake experiments after 4 h whereas different metabolites were detected. Furthermore, the quercetin-cinnamic acid amide showed pronounced activity in an in vivo AD mouse model at a remarkably low dose of 0.3 mg/kg.

Simplification of pharmacopoeial liquid chromatography methods for related substances of statins by hyphenated ultraviolet and charged aerosol detection.

For a more comprehensive characterization of a drug substance and its impurities, multidetector approaches are a helpful tool in liquid chromatography. In particular, the relatively inexpensive hyphenation of the ultraviolet (UV) with the charged aerosol detector (CAD) extends the scope from UV-active to non- or weak chromophore analytes, respectively. In this study, the chromatographic methods of the test for related substances of simvastatin and lovastatin in the European Pharmacopoeia were adapted to UV-CAD and thus allowing a more sophisticated detection of the weak chromophore dihydro impurity besides the other UV-active impurities. The compendial gradient program for simvastatin had to be modified (lowered initial acetonitrile percentage and increased gradient slope) because an additional critical peak pair emerged with the Hypersil C18 BDS column used here. Therefore, a Plackett-Burman design with 11 factors (including 4 dummy factors) was chosen to evaluate robustness of the adapted method. The flow rate, initial acetonitrile percentage, and column temperature were identified as three critical parameters that had to be carefully observed. Finally, the validity of the method for simultaneous detection of dihydrosimvastatin with CAD and of lovastatin and simvastatin as examples of UV detection was verified according to ICH Q2 (R1). In the case of lovastatin, the direct comparison with the pharmacopoeial method reveal that a determination with CAD is the more sensitive method.

Identification of low-level impurities in drug prototypes of carbocisteine by means of liquid chromatography-high-resolution mass spectrometry and general unknown comparative screening.

High-resolution tandem quadrupole time-of-flight mass analysers enable new automated workflows for untargeted data evaluation of complex samples like drug products. An example of such procedure is the so-called general unknown comparative screening (GUCS), which is used for software-assisted, automated identification of components that are only present in a sample and not in a reference. The GUCS approach has been employed for the first time to detect both degradation products and reaction products in drug products. Two different carbocisteine containing syrup prototypes - one with sucrose and the other with artificial sweeteners - were selected as examples after nine months of storage at 40 °C and 75% relative humidity. The samples were analysed chromatographically using a Coresep SB mixed-mode column and high-resolution MS and MS/MS data were recorded in information dependant acquisition mode on a Sciex X500R quadrupole time-of-flight mass spectrometer. Data analysis was considerably facilitated using the corresponding placebo formulation as reference samples. With the GUCS approach two hitherto unknown degradation products of carbocisteine, i.e. the carbocisteine lactam of the sulfoxides and the disulfide between l-cysteine and thioglycolic acid, were detected at low concentrations in both of the syrup formulations. The presumed structures were confirmed by in silico analysis of the fragment spectra and high-resolution LC-MS experiments with reference substances. Two additional impurities were found in the sucrose-containing sample and identified as the N-glycosides of carbocisteine and its lactam, respectively, using binary mixtures with a 13C-labelled monosaccharide.

Analytical Quality by Design: Achieving Robustness of an LC-CAD Method for the Analysis of Non-Volatile Fatty Acids.

An alternative to the time-consuming and error-prone pharmacopoeial gas chromatography method for the analysis of fatty acids (FAs) is urgently needed. The objective was therefore to propose a robust liquid chromatography method with charged aerosol detection for the analysis of polysorbate 80 (PS80) and magnesium stearate. FAs with different numbers of carbon atoms in the chain necessitated the use of a gradient method with a Hypersil Gold C18 column and acetonitrile as organic modifier. The risk-based Analytical Quality by Design approach was applied to define the Method Operable Design Region (MODR). Formic acid concentration, initial and final percentages of acetonitrile, gradient elution time, column temperature, and mobile phase flow rate were identified as critical method parameters (CMPs). The initial and final percentages of acetonitrile were fixed while the remaining CMPs were fine-tuned using response surface methodology. Critical method attributes included the baseline separation of adjacent peaks (α-linolenic and myristic acid, and oleic and petroselinic acid) and the retention factor of the last compound eluted, stearic acid. The MODR was calculated by Monte Carlo simulations with a probability equal or greater than 90%. Finally, the column temperature was set at 33 °C, the flow rate was 0.575 mL/min, and acetonitrile linearly increased from 70 to 80% (v/v) within 14.2 min.