Blackcurrant (Ribes nigrum L.) is a fruit rich in vitamins, fatty acids, minerals, essential oils and phenolic compounds, including anthocyanins. In the present work, two anthocyanin extraction methods from blackcurrant samples based on Ultrasound-Assisted Extraction (UAE) and Enzyme-Assisted Extraction (EAE) have been developed. A Plackett-Burman design with seven variables has been preliminary used for both UAE and EAE in order to determine the most influential variables in each methodology. After that, a Box-Behnken design was employed to optimize the extraction methods. The composition of the extraction solvent (% EtOH in water) has been the most influential variable for both UAE and EAE. The optimal extraction times have been 5 min for UAE and 10 min for EAE. No differences have been observed in anthocyanin extraction with both methodologies. Both methods have been applied to blackcurrant-derived products and proven their suitability for quality control analysis.

The aim of this study was to optimize the process of tablets compression and identification of film-coating critical process parameters (CPPs) affecting critical quality attributes (CQAs) using quality by design (QbD) approach. Design of experiment (DOE) and regression methods were employed to investigate hardness, disintegration time, and thickness of uncoated tablets depending on slugging and tableting compression force (CPPs). Plackett-Burman experimental design was applied to identify critical coating process parameters among selected ones that is: drying and preheating time, atomization air pressure, spray rate, air volume, inlet air temperature, and drum pressure that may influence the hardness and disintegration time of coated tablets. As a result of the research, design space was established to facilitate an in-depth understanding of existing relationship between CPPs and CQAs of intermediate product (uncoated tablets). Screening revealed that spray rate and inlet air temperature are two most important factors that affect the hardness of coated tablets. Simultaneously, none of the tested coating factors have influence on disintegration time. The observation was confirmed by conducting film coating of pilot size batches.

Faecal glucocorticoid analysis is a powerful non-invasive tool for the study of the animal endocrine status and stress physiology, which is mainly carried out by immunoassays, characterised by some limitations. In this study, an ultra high-performance liquid chromatography coupled to high resolution Orbitrap mass spectrometry (U-HPLC-HRMS) method was developed to confirm the presence of glucocorticoids in bovine faeces during a long-term stability study. Because of the complex nature of faeces, an appropriate extraction and purification procedure was developed. To this extent, a Plackett-Burman experimental design was successfully applied to determine the key conditions for optimal extraction of glucocorticoids from faeces. The targeted analysis, including natural and synthetic glucocorticoids, was successfully validated according to CD 2002/657/EC. Decision limits and detection capabilities for prednisolone, prednisone, methylprednisolone and the metabolites 20α-dihydroprednisolone and 20β-dihydroprednisolone ranged, respectively, from 0.15 to 2.95 μg kg(-1) and from 0.40 to 5.20 μg kg(-1). Limits of detection and limits of quantification for the natural glucocorticoids dihydrocortisone, cortisol and cortisone ranged, respectively, from 0.55 to 2.10 μg kg(-1) and from 0.70 to 5.00 μg kg(-1). The stability study of glucocorticoids in faecal matrix demonstrated that lyophilising the faeces, storage at -80°C, and aerobic conditions were optimal for preservation and able to significantly (p < 0.05) limit degradation up to 10 weeks.

Due to their growth-promoting effects, the use of synthetic glucocorticoids is strictly regulated in the European Union (Council Directive 2003/74/EC). In the frame of the national control plans, which should ensure the absence of residues in food products of animal origin, in recent years, a higher frequency of prednisolone positive bovine urines has been observed. This has raised questions with respect to the stability of natural corticoids in the respective urine samples and their potential to be transformed into synthetic analogs. In this study, a ultra high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) methodology was developed to examine the stability of glucocorticoids in bovine urine under various storage conditions (up to 20 weeks) and to define suitable conditions for sample handling and storage, using an Orbitrap Exactive™. To this end, an extraction procedure was optimized using a Plackett-Burman experimental design to determine the key conditions for optimal extraction of glucocorticoids from urine. Next, the analytical method was successfully validated according to the guidelines of CD 2002/657/EC. Decision limits and detection capabilities for prednisolone, prednisone and methylprednisolone ranged, respectively, from 0.1 to 0.5μgL(-1) and from 0.3 to 0.8μgL(-1). For the natural glucocorticoids limits of detection and limits of quantification for dihydrocortisone, cortisol and cortisone ranged, respectively, from 0.1 to 0.2μgL(-1) and from 0.3 to 0.8μgL(-1). The stability study demonstrated that filter-sterilization of urine, storage at -80°C, and acidic conditions (pH 3) were optimal for preservation of glucocorticoids in urine and able to significantly limit degradation up to 20 weeks.