July 2

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).

Molnár-Institute for Applied Chromatography, Schneeglöckchenstr. 47, 10407 Berlin, Germany, This email address is being protected from spambots. You need JavaScript enabled to view it.

Last year the Nobel Prize for Chemistry was granted to scientists, who were modeling physico-chemical interactions in biochemistry. Although we are modeling since 1985, the recognition of the importance of modeling for the whole Life Science is a great motivation for the Molnar-Institute to continue in finding the best column and the best separation in HPLC, by modeling a large number of possible chromatograms.

It is 40 years ago, that Csaba Horváth discovered in 1975 the reason for the success of Reversed Phase Chromatography at Yale – changes in the structure of water as part of the eluent. Complex calculations were required, but computers were just starting to emerge. He used a PDP11 “cabin” to describe, how acids dissociation might be modeled by equations.

Today we are modeling separation choices in a multi-variable Design Space to the most robust method for industrial drug production. With a computer generated separation model at hand, one can calculate the influence of various experimental parameters on the separation and, therefore, also model the robustness of a separation. This is extremely useful if a method should be used for a long time where one has to take into account day-to-day variations in method conditions.

The talk will demonstrate on case studies, how modeling software can work together with modern instruments to produce the best separation instead of what used to take months, in as fast as a few hours.

1 Restek Corporation, weerhaan 9, 4336KT, Middelburg, The Netherlands

2 Restek Corporation, PA, USA, This email address is being protected from spambots. You need JavaScript enabled to view it.

For several reasons, there is interest to replace helium for a different carrier gas in gas chromatography. Hydrogen is the obvious choice, but there is a concern on safety and reactivity. It is also possible to use nitrogen, but this is often not considered because it has a low optimal flow and velocity.

By using method translation and chromatogram modeling it became clear that the loss of efficiency using nitrogen could be perfectly compensated by using a smaller ID capillary of a shorter length. By replacing a 30m x 0.25 for a 20m x 0.15mm, it is demonstrated that separations under nitrogen are exactly the same as obtained with Helium, in the same analysis time, while using exact the same conditions for oven programming. Even the inlet pressures are very close.

The only price that has to be paid, is a loss in loadability, which means that this concept will not work for every application, but for many, it will. Besides the guaranteed availability, using nitrogen offers a big advantage in the cost and consumption volume of carrier gas, meaning cost per analysis will also benefit significantly.

Department of Chemistry, University of Parma, Parco Area delle Scienze 17/A, 43124 Parma, Italy, This email address is being protected from spambots. You need JavaScript enabled to view it.

Food products may come in contact with polymeric materials during processing by kitchen equipment and tableware, and storage and/or heating in plastic containers. This contact can be responsible for the migration of several compounds depending on food and plastic chemical properties, temperature and time of contact.

Polycarbonate is one of the most studied food contact material; migration of its monomer bisphenol A, and additives such as UV-absorbers or antioxidants, employed to prevent polymer degradation, has been reported to occur in food simulants (1). Ageing and light exposure are responsible for polymer degradation; besides, mechanical or chemical abrasion occurring during use and/or washing can damage the surface leading to release of oligomers and genesis of degradation products belonging to the so-called non-intentionally added substances (NIAS).

In this context, the development of suitable analytical methods to identify and to determine even low concentrations of possible contaminants is essential for safety assessment. High resolution and accurate mass spectrometry has been demonstrated to be a very promising technique in this field (2). The present study was aimed at developing and validating a method based on UHPLC coupled to Orbitrap technology to be employed for the identification and the monitoring of several compounds through a both targeted and untargeted approach. The investigation was focused on tableware of different age. Qualitative and quantitative analysis of bisphenol A and common additives employed in plastic production was carried out. Besides, the identification of colouring agents and polymer products of degradation was performed by means of the high mass accuracy. Statistical analysis permitted to correlate age, degree of surface damage, and bisphenol A migration, and allowed to discriminate new and old samples by evaluating the occurrence of a different pattern of degradation products.

References

1. Gao Y, Gu Y, Wei Y (2011) J Agric Food Chem 59, 12982.
2. Bignardi C, Cavazza A, Corradini C, Salvadeo P (2014) J Chromatogr A 1372, 133.

Institute of Chemistry, University of Silesia, 9 Szkolna Street, 40-006 Katowice, Poland, This email address is being protected from spambots. You need JavaScript enabled to view it.

Nowadays, protein and peptide analysis is on top of the research agenda of molecular biochemists, neurologists, geneticists, etc. At the same time, the enantioseparation of D/L amino acid pairs is on the one hand still a challenge and on the other, it is widely neglected. Consequently, a considerable confusion exists in various different areas of life sciences related to such issues, as homochirality of mammalian and human amino acids, geochronology making use of the amino acid clock, etc. Amino acid analysis with use of GC needs pre-column derivatization of amino acids to enhance the volatility thereof. Depending on pre-column derivatization type performed, the GC enantioseparation of amino acid pairs can be done with use of commercially available non-chiral or chiral capillary columns (i.e., in the so-called indirect or direct approach). The HPLC enantioseparation of amino acids is done mostly with use of regular non-chiral chromatographic columns (e.g., packed with C18, C8, etc.), yet upon pre-column amino acid derivatization with the Marfey’s reagent (or its analogues), to obtain respective diastereomers. For direct enantioseparation of underivatized amino acids, an absolutely limited spectrum of commercially available chiral HPLC columns is available from the market.

TLC is a flexible analytical tool which permits direct enantioseparation of amino acids, without a need to derivatize them prior to the analysis proper. Apart from commercial stationary phases used for direct enantioseparation of amino acids, a possibility exists of an in-home preparation of different chiral stationary phases and of specifically modified mobile phases which facilitate such enantioseparation. In most cases, molecular mechanism of direct amino acid enantioseparations by means of TLC is based on complexation of transition metals. Special attention will be given in this talk to selected examples of successful amino acid enantioseparations with use of TLC. In conclusion, on certain occasions direct enantioseparation of amino acids performed with aid of TLC can provide unequivocal results in shorter time and at a lower cost than the fully instrumental GC and HPLC technique.

 1 Irish Separation Science Cluster (ISSC), Department of Chemistry and the Analytical &Biological Chemistry Research Facility (ABCRF), University College Cork, Western Road, Cork, Ireland.

2 Biotechnology Research Institute, National Research Council Canada, Montreal, Quebec, Canada H4P2R2, This email address is being protected from spambots. You need JavaScript enabled to view it.

Increasingly nanoscale particles, pores and surfaces, are being tailor-made to provide high performance separation and detection. New superficially porous (core shell) silica particles for liquid chromatography are a good example in separation science, where nanoscale control over shell thickness enhances column efficiency. Nanoparticle incorporation in the separation and/or sensing roles, can also be an attractive means of enhancing selectivity and detectability.

In this work, sensitive and selective detection of disease indicating biomarkers is demonstrated using capillary electrophoresis with embedded gold and silica nanoparticles and amperometric detection at a boron doped diamond (BDD) electrode. The BDD electrode has emerged to play an important role in improving sensitivity in electrochemical detection. Luong et al.1 reviewed the application of the BDD electrode for sensitive detection in bioanalysis. Chen et al.2 reviewed the application of the BDD electrode for electrochemical detection in capillary electrophoresis.

By coating the fused silica capillary with gold nanoparticles (AuNPs) embedded in a cationic polymer poly (diallyldimethylammonium) chloride (PDDA), efficient separation and sensitive detection was achieved for analysis of 3-indoxyl sulphate (IXS), vanillylmandelic acid (VMA), homovanillic acid (HVA) and tryptophan (TRP) in urine3. The application of this separation and sensing approach is further applied in the field of bacterial cell-to-cell communication in the analysis of cell signalling molecules in the Pseudomonas Quinolone Signal (PQS) quorum sensing system4. P. aeruginosa is best known as an antibiotic resistant human pathogen associated with hospital-acquired infections and is the primary cause of morbidity and mortality in cystic fibrosis sufferers.

References:

1. J.H.T. Luong, K.B. Male, J.D. Glennon, Analyst, 2009, 134, 1965.
2. G. Chen, Talanta, 2007, 74, 326.
3. L. Zhou, J.D. Glennon, J.H.T. Luong, Anal Chem, 2010, 82, 6895.
4. L. Zhou, J.D. Glennon, F.J. Reen, F. O’Gara, C.M. McSweeney, G.P. McGlacken, Chem Comm, 2011, 47, 10347.

 1 Department of Chemistry, Laboratory of Analytical Chemistry, P.O. Box 55, 00014 University of Helsinki, Finland

2 Department of Engineering and Chemical Sciences, Karlstad University, 651 88 Karlstad, Sweden

3 Wihuri Research Institute, Haartmaninkatu 8, FIN-00290 Helsinki, Finland

4 National Institute for Health and Welfare, Public Health Genomics Unit, Biomedicum, Haartmaninkatu 8, FI-00251, Helsinki, Finland, This email address is being protected from spambots. You need JavaScript enabled to view it.

Although microsystems and miniaturized instrumental techniques, which can be considered key technologies for future progress in biochemistry, biotechnology, and medicine, have inspired many scientists toward further methodological developments, there continues to be a great need for analytical systems that enable dynamic and real-time monitoring of biological processes. Among many instrumental techniques and tools employed in exploring the role of biomolecular interactions in physiological and pathological phenomena, biosensors, capillary electromigration techniques, nano liquid chromatography and microscale thermophoresis enable small sample size, low reagent consumption, and label-free analysis, all of great advantage relative to more traditional approaches such as ELISA and affinity chromatography. The strength of the interactions is determined as affinity constants, partition coefficients, retention factors, and reduced mobilities.

In this talk versatility of capillary electromigration techniques, including adsorption energy calculations, that allow the differentiation of even small differences in the binding processes, will be introduced. Furthermore the exploitation of molecularly imprinted polymers in the isolation of human biomolecules will be demonstrated. Other techniques and approaches, such as microscale thermophoresis, quartz crystalline microbalance and molecular dynamics simulation calculations provide supportive and complementary insight into the interaction mechanisms.

[1] K. Lipponen, P. W. Stege, G. Cilpa, J. Samuelsson, T. Fornstedt, M.-L. Riekkola, Anal. Chem. 83 (2011) 6040.

[2] K. Lipponen, Y. Liu, P. Wanda Stege, K. Öörni, P. T. Kovanen, M.-L. Riekkola, Anal. Biochem.424 (2012) 71.

[3] K. Lipponen, S. Tähkä, M. Kostiainen, M.-L. Riekkola, Electrophoresis 35 (2013) 1106.

[4] K. Lipponen, S. Tähkä, J. Samuelsson, M. Jauhiainen, J. Metso, G. Cilpa-Karhu, T. Fornstedt, M. Kostiainen, M.-L. Riekkola, Anal. Bioanal. Chem. 406 (2014) 4137.

[5] J. Witos, J. Samuelsson, G. Cilpa-Karhu, J. Metso, M. Jauhiainen, M.-L. Riekkola,” Analyst, 2015, DOI: 10.1039/C5AN00210A.

 M. Curie-Skłodowska University, M. Curie-Skłodowska Sq.3, 20-031 Lublin, Poland, This email address is being protected from spambots. You need JavaScript enabled to view it.

Plants are unlimited source of biologically active substances used in the treatment of many human and animal diseases. They are also area of interest in searching new drugs that can be used to treat cardiovascular problems, diabetes, Alzheimer, cancer and other civilization illnesses. Because of increasing bacterial resistance against commonly used antibiotics there is also the urgent need for new antibacterials.

Chromatography is the most popular tool in the analysis of plant constituents. While high-performance liquid chromatography (HPLC) gives the biggest separation and detection possibilities, it does not provide information on biological properties of analytes. This problem can be solved by thin-layer chromatography –direct bioautography (TLC-DB) which can be used for searching biologically active substances in very complex matrices, such plants [1]. The principle of TLC-DB is that separation, biological detection (e.g. antimicrobial) and visualization are performed directly on a TLC layer [2]. In more detail, a developed TLC plate is dipped in a suspension of microorganisms growing in a nutrient broth and incubated in a humid atmosphere. The microorganisms grow directly on the surface of a TLC plate excluding spots of antimicrobials. Visualization is usually carried out by spraying a plate with tetrazolium salt, such as MTT, which is converted by living bacteria into the purple formazan. Creamy spots appearing against a colored background, so-called inhibition zones, point to the presence of antimicrobial agents. TLC-DB was used as a bio-guiding method to point substances with antibacterial activity in Matricaria recutita L., Achillea millefolium L., Salvia officinalis L., Thymus vulgaris L., Chelidonium majus L. and Hypericum perforatum L. extracts. The targeted substances, found by TLC-DB in analytical scale, were isolated in bigger amounts using TLC semi-preparative fractionation and then subjected to LC-MS analysis for structural evaluation.

[1] Marston, A. J.Chromatogr. A 2011, 1218, 2676–2683

[2] Choma, I. M.; Jesionek, W. Effects-Directed Biological Detection: Bioautography. In Instrumental Thin-Layer Chromatography; Elsevier: Amsterdam, 2014

 JLU Giessen, Heinrich-Buff-Ring 26-32, 35392 Giessen, Germany, This email address is being protected from spambots. You need JavaScript enabled to view it.

On the one hand, questions such as “Which compounds of the thousands of compounds in a complex sample are effective?” can hardly be solved with a rational effort. On the other hand, direct bioautography, which could answer such a question, led to diffuse zones and was not convincing analysts. Recently, it was shown that sharply-bounded bioactive zones were available after several hours of incubation, and in combination with targeted mass spectrometry, led to a streamlined methodology that is able to answer such effect-directed questions within 15 min per sample [1-3].

The first part of the streamlined hyphenated method is an effect-directed screening of up to 22 raw extracts in parallel. Thus, this part (HPTLC-UV/Vis/FLD-(bio)assay) can be described as a non-targeted, effect-directed detection of single or also coeluting effective compounds of the complex sample. The second part is a highly targeted characterization of the discovered effective compounds via the direct link to structure elucidating techniques (HPTLC-HRMS or NMR or ATR FTIR). The zone of interest is selectively eluted via a short orthogonal column into the HRMS or into a microvial for NMR or ATR FTIR recordings. Thus, information on effective compounds in a complex sample and their sum formulae can be obtained from a single chromatographic run.

Depending on the bioassay or enzymatic assay selected, for example, œstrogens, antibiotics, xanthine oxidase inhibitors, acetylcholinesterase inhibitors or tyrosinase inhibitors are discovered in complex samples. However, every technique has its limitation, and for oxidation-prone or volatile compounds, this streamlined methodology is limited in the information content. Nevertheless, it may serve as a survey on effect-directed components in complex samples. Benefits may result from the side-by-side sample comparison, the matrix-tolerance, the avoidance of carry over and of discrimination, the always fresh adsorbent, the comparatively low-tech workflow as well as the multifold evaluation of the separated sample saved on the plate.

Literature:

[1] G.E. Morlock, ACS Syposium Series 1185 (2013) 101-121.

[2] G.E. Morlock, I. Klingelhöfer, Anal. Chem. 86 (2014) 8289–8295.

[3] I. Klingelhöfer, G.E. Morlock, J. Chromatogr. A 1360 (2014) 288-295.

 ¹ School of Pharmacy and Applied Science, La Trobe Institute of Molecular Sciences, La Trobe University, Edwards Rd, Bendigo 3550, Australia

² Physiology Department, Medical School, Universiti Teknologi Mara, Selangor, Malaysia, This email address is being protected from spambots. You need JavaScript enabled to view it.

This study describes a simple high performance thin layer chromatographic (HPTLC) method for the quantification of apigenin, chamazulene, bisabolol and parthenolide in leaf and flower head extracts from feverfew, German chamomile and marigold and comparison of their free radical scavenging activities. Feverfew (Tanacetum parthenium) leaves have been traditionally used in the treatment of migraine with parthenolide being the main bioactive compound. However, due to similar flowers, feverfew is sometimes mistaken for the German chamomile (Matricaria recutita). Bisabolol and chamazulene are the main components in chamomile essential oil. Marigold (Calendula officinalis) was included in the study for comparison, as it belongs to the same Asteraceae or Compositae (commonly known as daisy) family.

Parthenolide was found to be present in all leaf extracts but was not detected in calendula flower extract. Chamazulene and bisabolol were found to be present in higher concentrations in chamomile and Calendula flowers. Apigenin was detected and quantified only in chamomile extracts. Antioxidant activity in sample extracts was compared by superimposing the chromatograms obtained after post chromatographic derivatization with DPPH free radical and post chromatographic derivatization with anisaldehyde. It was found that extracts from chamomile flower heads and leaves have the most prominent antioxidant activity, with bisabolol and chamazulene being the most effective antioxidants.

 School of Pharmaceutical Sciences, EPGL, University of Geneva, University of Lausanne, 30, quai Ernest-Ansermet, CH-1211 Geneva, Switzerland, This email address is being protected from spambots. You need JavaScript enabled to view it.

Analysing metabolomes from various natural sources is a task that requires methods providing high chromatographic resolution for detailed metabolite profiling or high throughput for rapid fingerprinting for metabolomics [1,2]. Furthermore these methods should give on-line spectroscopic information for the identification of each individual metabolite for dereplication purposes. In this respect the introduction of UHPLC has allowed a remarkable decrease in analysis time and increase in peak capacity, sensitivity and reproducibility compared to conventional HPLC. In complement to this powerful chromatographic method, the introduction of benchtop high resolution MS instruments that provide sensitive detection and accurate MS and MS/MS information for dereplication has been key for metabolomics. Recently the development of neural network mining methods for metabolite identification have revolutionized conventional MS/MS database search for establishing link between metabolites having similar fragmentation pattern that greatly help metabolite annotation. For unambiguous de-novo identification, LC-MS targeted isolation together with at-line microNMR approaches provide low microgram level of metabolites sufficient for acquiring a full set of 1D and 2D NMR spectra. Such information complements well the search in MS and chemotaxonomic data bases for dereplication. In addition to these methods, HPLC activity-based profiling provides information on the bioactivity of metabolites of interest, directly at the analytical scale, speeding up the drug discovery process in natural product research. The impact of these technologies in NP research studies and future trends will be discussed. This will be illustrated by examples or our latest metabolomics and bioactivity-guided isolation studies on plants and microorganisms.

1. Wolfender JL, Rudaz S, Choi YH, Kim HK. Plant metabolomics: from holistic data to relevant biomarkers. Curr. Med. Chem. 2013 20: 1056-90.
2. Wolfender J-L, Marti G, Thomas A, Bertrand S. Current approaches and challenges for the metabolite profiling of complex natural extracts. J Chromatogr A 2015 1382: 136-164.

 1 National Research Council, Institute of Chemical Methodologies, Area della Ricerca di Roma 1, 00015 Monterotondo, Rome, Italy

2 Molnár-Institute Schneegloeckchenstrasse 47, 10407 Berlin, Germany, This email address is being protected from spambots. You need JavaScript enabled to view it.

Plant secondary metabolites are organic compounds produced by plants, which are not involved in the growth, photosynthesis, reproduction and other primary functions of the plants but are believed to have a role in defence mechanisms, such as protection against herbivores, pests and pathogens. Phenolic compounds are one of the main classes of secondary metabolites and because are widely distributed in the plant kingdom form an integral part of human diet. In addition to confer specific sensorial characteristics to plant-derived foods and/or beneficial effects on human health, phenolic compounds are also biomolecules with pharmacological activity employed in phytotherapeutic medicine. The identification and quantification of these target compounds in plants tissues and agro-food matrices is a challenging task, continuously requesting the development of more robust, efficient and sensitive instrumental analytical techniques. This communication discusses fundamental and practical aspects of both reversed phase high performance liquid chromatography (RP-HPLC) and capillary zone electrophoresis (CZE) tailored for the analysis of plant secondary metabolites. The communication reports the results of our recent studies carried out to evaluate the influence of various operational parameters and experimental conditions employed in CZE and in RP-HPLC on the separation performance of phenolic compounds and other secondary metabolites in plant tissues and agro-food matrices. Because both RP-HPLC and CZE require the use of a liquid phase, whose composition plays a fundamental role on the separation mechanisms and performance, most of our investigations has been focused on the selection of the proper composition of the liquid phase, which, in the case of RP-HPLC, has been optimized by a quality by design approach.

 The Weizmann Institute of Science. PO.BOX 76100, Rehovot, Israel, This email address is being protected from spambots. You need JavaScript enabled to view it.

Metabolomics and Lipidomics, involve targeted and untargeted analysis of various endogenous biomolecules, in complex samples estimated 5000-20,000 metabolites in a single plant. Thus multiple modes of analysis are needed to enable identification as well as quantitation to get a better understanding of these thousands Metabolites.

NMR, GC/MS, UHPLC/MS, USFC/MS are currently used for metabolome analysis, lately advancements in Ion Mobility separation, (a gas-phase separation tool) namely Traveling-Wave Ion Mobility (TWIM) combined with high-sensitivity mass spectrometry QTOF-MS, have been used to set the stage for a myriad of metabolomics studies, in plants and other organisms.

Here we describe the use of UHPLC - TWIM- QTOF-MS, to generated, Retention Times, Drift times, and accurate Mass. For the polar and semi polar metabolites. In addition, we used Super Critical Fluid Chromatography USFC-TWIM-QTOF-MS for the non-polar and semi polar metabolites analysis.

Plant and Algae samples data, were analyzed by a dedicated software tool, using multiple parameters such as, Retention Time, exact mass, MS/MS fragments, isotope distribution, and drift times, in order to achieve a three dimension resolution and more reliable data. That enables searching external databases, such as: LipidBlast, ChemSpider, METLIN, Elemental Composition, and our own database.

With the added parameters and the software tool, we found many more lipids in our samples, than with our previous method. We hope to verify some of these in the near future.

 1 University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, Department of Chemistry and Biochemistry, Mănăstur 3-5 Cluj-Napoca, 400372, Romania

2 Università degli Studi di Messina Dipartimento di Scienze dell'Ambiente, della Sicurezza, del Territorio, degli Alimenti e della Salute, Viale F. Stagno d'Alcontres, 3198166, Messina, Italy, This email address is being protected from spambots. You need JavaScript enabled to view it.

Fruits and berries contains large amount of xanthophylls which are often esterified with various fatty acids [1]. The saponification procedure, usually performed prior chromatographic separation, also removes the acyl moieties of xanthophyll esters and unable the proper characterization of native carotenoid extracts from plant materials.

The fruits of Hippophae rhamnoides (sea buckthorn), Physalis alkekengi (Chinese lanterns) and Prunus armeniaca (apricot) fruits were used for the analysis of esterified xanthophylls. Characterization of unsaponified extracts was performed by HPLC-DAD-APCI-MS on C18 and C30 RP columns. Pure esters of zeaxanthin (Z) and β-cryptoxanthin (BCR) with myristic (M), palmitic (P), oleic (O) and linoleic (L) acids were obtained by semi-synthesis and used for identification and stability studies. The major xanthophylls in all three fruits are Z and BCR, small amounts of lutein being identified only in sea buckthorn berries. In the sepals of Physalis alkekengi Z and β-cryptoxanthin was almost completely esterified with M and P acids. Sea buckthorn berries of various cultivars were analyzed and found to contain a significantly higher number (14) of both mono- and diesters. Z was mainly esterified with P acid but esters with O and palmitoleic acids were also found. Interestingly, lutein was the only xanthophyll esterified with polyunsaturated fatty acids (L, hexadecadienoic). Apricot fruits are rich sources of β-carotene and the only important xanthophyll is BCR. Investigations on several cultivars showed a high variability in carotenoid and esters content and profile. The major fatty acids esterifying BCR are P, stearic and lauric acids but small amounts of esters with O, L and linolenic acids were also detected. Stability tests performed on pure esters and on sea buckthorn fruits showed a better stability of esterified xanthophylls compared to the free form during thermal treatment and storage.

1. This work was supported by a grant of the Romanian National Authority for Scientific Research CNCS-UEFISCDI, project PN-II-ID-PCE-2011-3-0721

References

1. Weller, P., Breithaupt, D.E., J. Agric. Food Chem., 2003, 51, 7044-7049