Session 9

Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Belgrade, Vojvode Stepe 450, 11000, Belgrade, Serbia, This email address is being protected from spambots. You need JavaScript enabled to view it.

Blood-Brain Barrier (BBB) plays an essential role in protecting Central Nervous System (CNS) from harmful agents present in the bloodstream. In order to estimate central nervous delivery of compounds a lot of useful in vitro models have been developed. The most used are Parallel Artificial Membrane Permeability Assay (PAMPA)[1], Biopartitioning Micellar Chromatography (BMC) [2] and cell based assays. BMC is a mode of micellar liquid chromatography that uses micellar mobile phases of Brij35 in adequate experimental conditions. It can be useful in mimicking the drug partitioning process into biological systems, because the characteristics of BMC system are similar to biological barriers and extracellular fluids. In this work, the BBB penetration of α-adrenergic/imidazoline receptor ligands and related CNS active compounds were examined using BMC system. Obtained retention factors were correlated with permeability coefficients (logPe) obtained using PAMPA [3], as well as retention factors obtained using Reversed-Phase High-Performance Liquid Chromatography (RP HPLC). Further, Partial Least Square (PLS), Multiple Linear Regression (MLR) and Artificial Neural Networks (ANN) models were developed using retention data from BMC system and molecular parameters calculated for the optimized compounds using Chem3D Ultra 9.0.1, Dragon and ADMET predictor software. Formed models could be used as time and cost efficient screening tool for evaluation of blood-brain barrier permeation of novel α-adrenergic/imidazoline receptor ligands, as promising drug candidates for treatment of hypertension or neurological diseases.

Acknowledgment

This work was supported by the Ministry for Science of the Republic of Serbia (Contract No. 172033). The COST Actions CM1103 and CM1207 are also kindly acknowledged.

References

[1] Avdeef A, 2005. The rise of PAMPA. Expert Opin. Drug Metab. Toxicol. 1, 325-342

[2] Molero-Monfort M, et al., 2000. Micellar liquid chromatography for prediction of drug transport. J. Chromatogr. A 870, 1-11

[3] Vucicevic J, et al., 2015. Prediction of BBB permeation of α-adrenergic and imidazoline receptor ligands using PAMPA technique and QSPR analysis. Eur. J. Pharm. Sci. 68, 94-105

1 Laboratory of Pharmaceutical Quality Control-UFRGS, Av. Ipiranga 2752, 90610000, Porto Alegre, Brazil

2 Faculty of Pharmacy-UFRJ, Av. do Aloízio, 27930560, Macaé, Brazil, This email address is being protected from spambots. You need JavaScript enabled to view it.

Identification of pharmaceutical degradation products (DP) used to be mostly performed by tiring and time-consuming separation/isolation techniques (e.g. preparative column and thin layer chromatography), followed by purification processes, and finally analysed by spectroscopic methods. Development of more sensitive and precise mass analysers and improvement of liquid chromatography coupled to mass spectrometry (LC-MS), outcome on elimination of isolation and fraction collection steps from DPs characterization process. Atmospheric pressure ionization, especially electrospray ionization (ESI), is the leading nebulizing method in analysis of pharmaceutical drugs. Several methods have been developed with ion trap as mass analyser but most recently, quadrupole time-of-flight has been widely employed to analyse DP mass fragments due its high resolution and measurements accuracy. This work aims to review later trends regarding characterization and identification of drug impurities upcoming from degradation processes using LC-MS, practical approach will be set throughout the presentation of studies performed by our research group. Delapril (DEL) and manidipine (MAN) hold synergic antihypertensive effects. DEL (alkaline medium) and MAN (UVA radiation) exposed to stress conditions presented three and two DPs, respectively. Identification analyses were conducted on a LC-ESI triple quadrupole MS. Another combined drug formulation studied was vildagliptine-metformine, type 2 Diabetes manager. Due to divergent physicochemical properties, two separation techniques had to be performed (reverse phase and HILIC columns) in order to identify the main DP of each compound. Anticoagulant rivaroxaban (RIV) under stress state yield emerge of three DP, although they had different structures, m/z values, and retention times, it was possible to find similarity between fragmentation patterns. LC-MS/MS technique allows reliable drug DP analysis in short time and with less chemicals, therefore reducing costs and environmental exposition. DP elucidation enables major access and knowledge as to drug stability, increasing quality control tools and safer treatments.

Acknowledgment: PPGCF-UFRGS.

Technische Univesrität München, Chair of Urban Water Systems Engineering, Am Coulombwall 8, 85748, Garching Munich, Germany, This email address is being protected from spambots. You need JavaScript enabled to view it.

Most environmental and food samples have complex matrices and contain compounds with a wide range of polarity.

Reversed phase liquid chromatography (RPLC) has been established and frequently used for the separation of various apolar (logD>1) and mildly polar (logD>0) compounds.

On the other hand, very polar compounds (logD<0) can effectively be separated by Hydrophilic interaction liquid chromatography (HILIC). This technique became more convenient since the mechanisms have been understood better. The use of either RPLC or HILIC separation technique in compound screening, generally results in a loss of a large fraction of the analytes present to a sample. The serial on-line combination of HILIC and RPLC lead to an easy, robust and relatively fast screening technique, which combined with atmospheric pressure ionization (API) and mass spectrometry (MS) constitute a unique tool in the monitoring of real samples [1-3].

The successful analysis of phenols in wine will be presented as an example for the wide separation ability of the HILIC-RP coupling [1], followed by some screening results of environmental interest. We present for the first time new impressive projects with RPLC-HILIC-API-MS resulting in highly informative screening details over a broad range of polarity.

[1] G. Greco, S. Grosse, and T. Letzel: HILIC-RP HPLC-API-ToF MS for the determination of polar and apolar organic molecules in wines. Journal of Separation Science 2013, 36 (8), 1279-1388.

[2] G. Greco and T. Letzel: The hyphenation of HILIC, RP- HPLC and API – MS. LCGC N. America and Spectroscopy supplement: Current Trends in Mass Spectrometry 2012, May, 40-44.

[3] G. Greco, S. Grosse, T. Letzel: Robustness of a method based on the serial coupling of reversed-phase and zwitterionic hydrophilic interaction LC/MS for the analysis of phenols. Journal of Separation Sciences 2014, 37 (6), 630-634.

1 “Scienze del Farmaco e Prodotti per la Salute” Department, University of Messina, viale Annunziata, 98168 – Messina, Italy

2 “Scienze dell’Ambiente, della Sicurezza, del Territorio, degli Alimenti e della Salute” Department, University of Messina, Viale F. Stagno d’Alcontres 31, 98166 Messina, Italy

3 Chromaleont s.r.l., c/o “Scienze del Farmaco e Prodotti per la Salute” Department, University of Messina, viale Annunziata, 98168 – Messina, Italy

4 University Campus Bio-Medico of Rome, Via Álvaro del Portillo, 21 - 00128 Rome, Italy", This email address is being protected from spambots. You need JavaScript enabled to view it.

Lipidomics is a branch of metabolomics and aims to study all the lipids within a living system or in complex biological samples. The abundance and differences of individual lipid molecular species in biological samples may be indicative as a clinical tool for risk assessment and disease monitoring. In this study, we developed a practical workflow for lipid profiling of biological samples by using several analytical techniques such as one- and multidimensional chromatography coupled to mass spectrometry (MS). Specifically, liquid chromatography (LC) separation is necessary for preliminary identification of lipid molecular species, because it can increase sensitivity for low abundant analytes by separating them from matrix components which might produce ion suppression; moreover, it can provide more detailed information on lipid isomers compared to direct infusion mass spectrometry. Unlike gas chromatography (GC), LC techniques offer a wide variety of separation mechanisms, thanks to a higher number of stationary phases. Different approaches in multidimensional liquid chromatography were developed for an increased resolving power compared to one-dimensional methods. The analysis of intact lipid constituents was carried out with both hydrophilic interaction liquid chromatography (HILIC) mode (for a class-type separation) and reversed-phase (RP) mode (for lipid species separation). GC analyses provided information on fatty acid profile of biological samples investigated (whole blood, plasma, erythrocyte membrane, muscle).

The main focus of this work is a lipidomic study of different biological samples, in order to perform a full characterization of lipid molecular species.

1 University of Florence, Via della Lastruccia 3 - 13, 50019 Sesto Fiorentino (Florence), Italy

2 ABSciex, Viale Lombardia, 218, 20047 Brugherio, Monza Brianza, Italy

3 Department of Plant, Soil and Environmental Science, Viale delle Idee, 30-50019 Sesto Fiorentino, Florence, Italy

4 Department of Chemistry, Via Pietro Giuria 5, 10125 Turin, Italy, This email address is being protected from spambots. You need JavaScript enabled to view it.

Vaccinium myrtillus berries (wild bilberry) are an important economic resource in various European regions, including Italian mountain areas, owing to their use as supplement of anthocyanins in the human diet. Berry anthocyanins are in fact recognized as anti-allergic, anti-inflammatory, antihypertensive, antimicrobial and anticancer molecules [1]. Recently, the presence of a different Vaccinium species, namely V. gaultherioides, has been increasingly observed in the zones traditionally populated by V. myrtillus, such as Tuscan Apennines. The phenotype of V. gaultherioides berry is very similar to the one of V. myrtillus and the two berries can be confused by the harvesters involved in the production chain of transformed bilberry. This aspect is potentially awkward since no data regarding the primary and secondary metabolic profiles of V. gaultherioides berries, including anthocyanins, are reported in literature.

For this reason, the polyphenolic composition of V. myrtillus and V. gaultherioides berries has been comparatively investigated using liquid chromatography coupled with mass spectrometry. Different stationary phases (e.g. C18 and pentafluorophenyl) and particle sizes (2.6 and 1.7 µm) have been tested in order to enhance the chromatographic selectivity. Target tandem and non-target high-resolution mass spectrometric approaches have been investigated.

Furthermore, antiradical (DPPH method) and antioxidant (FRAP method) activities have been determined for the two berry species. The results showed that the two species exhibit very different abundances of phenolic substances and antiradical/antioxidant activities. More in detail, V. myrtillus fruits had much higher contents of phenolic compounds and antiradical/antioxidant activities than those of V. gaultherioides. The anthocyanin profiles of the two species was different as well, being V. myrtillus and V. gaultherioides berries dominated by delphinidin/cyanidin glycosides, and by malvidin derivatives, respectively.

Based on these results, it is of paramount importance to carefully distinguish between the two species during the harvesting activities.

This research was funded by "PRAF 2012-2015, Misura 1.2. e)" of Regione Toscana.

Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i., Kralovopolska 135, 61265, Brno, Czech Republic, This email address is being protected from spambots. You need JavaScript enabled to view it.

DNA structure, its structural changes and interactions can be studied using DNA probes and markers, e. g. osmate adducts. Short synthetic oligodeoxyribonucleotides (ODNs) serve as model molecules for studies of properties of such adducts and of reactions leading to their formation [1].

For the research of ODNs derivatives it is important to study the mother ODNs first. From the existing literature (e.g. Refs. 2, 3) it is not possible to create any consistent notion of the electrophoretic behaviour of ODNs. At pH values 8 – 11, which dominate in CE separations of nucleotides and their oligomers, ODNs migrate anionically because of their surplus negative charge. This fact led to expectation that electrophoretic behaviour and properties of ODNs are determined exclusively by their negative electrophoretic charge.

The presented study was motivated by the expectation stated above. We investigated the influence of background electrolyte (BGE) pH, composition and concentration on the electrophoretic transport of short ODNs (mostly 5 – 10 nucleotide units long) in fused silica capillary. ODNs mobilities, which changed with changes in all three BGE characteristics, cannot be systematically explained even if the BGE characteristics in addition to acid-base properties of ODNs are considered. Even more complex are separation efficiency variations. Our experiments indicate that the electrophoretic properties and behaviour of short ODNs in aqueous solutions are more complex than it has been presented until now. Probably there are other processes relevant for CE separations of short ODNs. We show that separation efficiencies higher than 400 000 theoretical plates can be reached.

This work was supported by the Czech Science Foundation (grant P206/12/G151).

References:

1. Fojta M., Kostečka P., Pivoňková H., Horáková P., Havran L.: Curr. Anal. Chem. 7, 35 (2011).

2. Dolník V., Liu J., Banks F. B., Jr., Novotný M.V., Boček P.: J. Chromatogr. 480, 321 (1989).

3. McKeown A. P., Shaw N., Barrett D. A.:Electrophoresis 22, 1119 (2001).

Jagiellonian University, Department of Analytical Chemistry, Ingardena 3, 30-060 Krakow, Poland, This email address is being protected from spambots. You need JavaScript enabled to view it.

Ergot alkaloids are a group of ergoline derivatives, such as ergine (LSA), ergometrine, lysergic acid α-hydroxyethylamide (LSH), lysergol and others. Many of those substances exhibit biological activity in human, including psychedelic effects [1].

Ergot alkaloids are produced by Clavicipitaceae fungi and can be found in their plants hosts, including Convolvulaceae family, known under common name Morning Glory. Due to their hallucinogenic properties and wide, legal availability, Morning Glory cultivars are used recreationally, but with a risk of poisonings [2]. Therefore, the assessment of ergot alkaloids concentration in plants from the Convolvulaceae family is highly important for toxicological and forensic purposes.

A simple and rapid method has been developed for determination of ergine and ergometrine, and identification of other ergot alkaloids in Morning Glory seeds from as little as 10 mg of sample. For the purpose of the analysis, three extraction techniques: microwave-assisted extraction (MAE), ultrasound-assisted extraction in ultrasound bath (UAE-B) and sonotrode extraction (UAE-S) have been optimized in terms of time, temperature and solvent, using experimental design. Optimal conditions were established and it was assessed that UAE-B method was the most suitable, enabling complete extraction of ergine and ergometrine without degradation of labile analytes. The optimized extraction parameters were: 60°C, 30 min, in 10 mL of MeOH:water 7:3 (v:v). The extracts were filtrated and analysed directly by HPLC-Q-TOF-MS in scan mode for quantitative and MS/MS mode for qualitative analysis. The optimized UAE-B/LC-MS method was validated and applied for Morning Glory samples. Ergine and ergometrine were determined in samples of Ipomoea purpurea and “Morning Glory” cultivars; other alkaloids (such as LSH, chanoclavine, penniclavine, lysergol) have been identified.

[1] A. Paulke et al., “Identification of legal highs - Ergot alkaloid patterns in two Argyreia nervosa products”, Forensic Sci. Int., 242, 62–71, 2014.

[2] H. B. Klinke et al., “Two cases of lysergamide intoxication by ingestion of seeds from Hawaiian Baby Woodrose”, Forensic Sci. Int., 197, 1–5, 2010.

Department of Chemistry, Aristotle University of Thessaloniki, 54124, Thessaloniki, Greece, This email address is being protected from spambots. You need JavaScript enabled to view it.

In the present study we explore the capabilities of a universally available software, Microsoft Excel. We demonstrate that Microsoft Excel 2010 or 2013 can be used in the whole procedure of an optimization scheme in liquid chromatography. With our package we take the advantage of its use in the whole procedure of separation optimizations in HPLC for modelling multilinear gradient retention data obtained in single and double gradient elution mode by changing organic modifier(s) content and/or eluent pH. For this purpose, ten chromatographic models were used and four methods were adopted for their application. The methods were based on a) the analytical expression of the retention time, provided that this expression is available, b) the retention time estimated using the Nikitas-Pappa approach, c) the stepwise approximation, and d) a simple numerical approximation involving the trapezoid rule for integration of the fundamental equation for gradient elution. For all these methods, Excel VBA macros have been written and implemented using two different platforms; the fitting and the optimization platform. The fitting platform calculates not only the adjustable parameters of the chromatographic models, but also the accuracy of these parameters and furthermore predicts the analyte elution times. The optimization platform determines the gradient conditions that lead to the optimum separation of a mixture of analytes by using the Solver evolutionary mode, provided that proper constraints are set in order to obtain the optimum gradient profile in the minimum gradient time. Undoubtedly, this environment will be a powerful tool to the application of systematic optimization strategies by the plethora of chromatographers.

Acknowledgment

The project is realized in the framework of the Operational Program “Education and Lifelong learning” and is co-funded by the European Union (European Social Fund) and National Resources as part of the Grant Standardizing Metabolomics (Excellence II: Metabostandards 5204).