July 3

University of Zagreb, Faculty of Chemical Engineering and Technology, Marulićev trg 19, 10000 Zagreb, Croatia, This email address is being protected from spambots. You need JavaScript enabled to view it.

A priori knowledge of the retention time of a given analyte simplifies the determination of separation conditions therefore quantitative structure retention relationship (QSRR) modelling might be considered a reasonable selection. The first problem in QSRR modelling is to select the most informative descriptors from among a large number of mutually correlated descriptors, while the second one is to build the core model of isocratic and/or gradient elution retention. A lot of conventional methods have been elaborated that are mainly based on different types of regression and simple variable selection methodology (i.e. principal component analysis), showing rather questionable prediction ability. This work reveals recent results on development of artificial intelligence (AI) hybrid methodology implementing all three AI paradigms: artificial neural networks, genetic algorithms and fuzzy logic. The developed models were fully optimized and validated with external set of compounds showing significant improvement of generalization ability. Furthermore, recent demands for increasing the productivity using the gradients, in combination with ever growing complexity of analyzed samples, are introducing an additional request on the analytical system – beside being fairly separated, the peaks are required be as “smoothly” shaped as possible to ensure their precise quantification. In other words, the analysts are becoming interested in peak shapes and peak shape modelling as well. This work also discusses recent developments is peak shape modelling based on QSRR modelling. The developed models are based on generalized logistic distribution and hybrid AI systems. The external validation results show promising predictive ability, but still indicate that there is much to be done before QSRR based optimization strategy could be efficiently built into a useful commercial software.

1 Thermo Fisher Scientific GmbH, Im Steingrund 4-6, D-63303 Dreieich

2 Leopold-Franzens University Innsbruck, Innrain 80-82, A-6020 Innsbruck, This email address is being protected from spambots. You need JavaScript enabled to view it.

HPLC applications based on capillary columns have seen an impressive progress in the last decade. As an analytical tool capillary HPLC offers the following advantages:

• Easier hyphenation with mass spectrometers
• Significant reduction of eluent consumption
• Higher mass sensitivity in comparison to conventional HPLC
• Ideal for applications with limited sample volume

Introduced in 2010, capillary ion chromatography with electrolytic eluent generation offers similar advantages, although the advantages listed above have a different significance for ion chromatography applications. The most significant advantage of ion chromatography in the capillary format is the marked reduction of eluent consumption, which allows continuous operation over a long period of time. In conventional water analysis, sample volume is usually not the issue, but users investigating corrosion phenomena or metabolic pathways are benefitting from much smaller sample volumes (<< 1 µL) used in capillary IC. The higher mass sensitivity in capillary IC in comparison to conventional IC has a huge impact on trace analysis, because the same concentration sensitivity can be achieved with much smaller sample volumes. Last but not least, capillary IC operating with very small flow rates improves the compatibility with modern mass spectrometers.

The expanded pressure tolerance of electrolytic eluent generation in capillary IC systems up to 34.5 MPa (5000 psi) allows the use of higher linear velocities of the mobile phase and thus much faster analysis times. On the other hand, it also facilitates high-resolution separations of complex samples through the use of packing materials with smaller particle sizes. In this presentation, examples of high-throughput and high-resolution separations of inorganic and organic ions from different application areas will be shown, utilizing resin-based as well as monolithic stationary phases.

Chair of Environmental Chemistry and Bioanalytics, Faculty of Chemistry & Centre for Modern Interdyscyplinary Technology Nicolaus Copernicus University, Gagarin 7, 87-100 Toruń, Poland, This email address is being protected from spambots. You need JavaScript enabled to view it.

Rapid detection and identification of microorganisms is a challenging and important aspect in many areas of our life, beginning with medicine, ending with industry. Unfortunately, classical methods of microorganisms’ identification are based on time-consuming and labor-intensive approaches. Screening techniques require rapid and cheap grouping of bacterial isolates, however modern bioanalytics demands comprehensive bacterial studies on molecular level. The new approach to the rapid identification of bacteria is to use the electromigration techniques, especially capillary zone electrophoresis (CZE). CZE is an important technique used in the analysis of microorganisms. However, the analysis of microbial complexes using this technology still encounters several problems – uncontrolled aggregation and/or adhesion to the capillary surface. One way to resolve this issue is the CZE analysis of microbial cell with surface charge modification by bivalent metal ions (e.g. Ca2+aq, Zn2+aq). Under the above conditions, bacterial cells create compact aggregates, and fewer high-intensity signals are observed in electropherograms. The capillary electrophoresis of microbial aggregates approach with UV and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) detection has been presented.

The aim of this study was to characterize the charge and surface of the bacteria in order to determine their role in adhesion and aggregation phenomena during the electrophoretic separation. The use of experimental techniques, including electrochemical and electrophoretic allowed the description of the relationship between the acid-base properties of pathogens and their behavior. In this studies was performed the identification of bacterial via spectrometric techniques.

Acknowledgments:

This work was supported by the National Science Centre (NCN, Warsaw, Poland) projects No. 2013/08/W/NZ8/701 – Symfonia-1, Maestro-6 (2015-2017) and by European Social Found, Polish National Budget, Kuyavian-Pomeranian Voivodeship Budget (within Sectoral Operational Programme Human Resources) “Krok w przyszłość 2014-2015”

Department of Pharmaceutics, Semmelweis University, Hőgyes Endre St. 7., H-1092, Budapest, Hungary, This email address is being protected from spambots. You need JavaScript enabled to view it.

The lecture sums up the modern tools of the radiochromatography in preclinical and clinical pharmacokinetic and drug metabolism research. The essential pharmacokinetic and drug metabolism information of different species (mouse, rat, dog, rabbit and human) contribute to the final drug registration process.

The high sensitivity (pg/ml, fg/ml, at/ml) and highly selective hyphenated techniques (LC/Triple Quad-Jet Stream-ESI-MS and GC/MS-MS, etc.) required for the pharmacokinetic studies had replaced the conventional methods of detections such as GC and HPLC.

Nowadays in the course of drug development the radioactive isotopes (beta and gamma single and/or double source) labeled (³H, ¹⁴C, ⁹⁹Tc, ¹³¹I) pharmacokinetic studies combined with the new generation of triple-quad MS techniques (LC, CE, OPLC) are essential. A number of related case studies will be presented.

The former Imaging Techniques (DAR, PIT) and the new generation of in vitro – in vivo Imaging Techniques (MALDI Imaging, nanoScan, PET/MRI in animal and human studies) will also be presented.

A complex multi-step process will be illustrated from separation, purification, isolation to structure elucidation (GC-MS, LC-MS/MS, LC-NMR) of minor and major metabolites derived from animal and human biological matrices. The addition of the above systems to the off-line and on-line separation and radioactivity detection possibilities of OPLC-DAR/PIT, OPLC-RD, HPTLC-DAR-MS and GC-RD, HPLC-RD and the combined multi hyphenated techniques, OPLC-DAD-RD-MS/MS, OPLC-DAD-RD-NMR as well as LC-DAD-RD-MS/MS and LC-DAD-RD-NMR resulted in a new, flexible and rapid high-performance complex solution in the metabolism research.

1 University of Castilla-La Mancha, Campus Universitario s/n, 02071, Albacete, Spain

2 Spanish Agency for Health Protection in Sport. Calle Greco s/n. 28040 Madrid. Spain, This email address is being protected from spambots. You need JavaScript enabled to view it.

Boldenone (androsta–1,4–dien–17β–ol–3–one, Bo) is an anabolic steroid known to have been used in cattle breeding or equine sport as a doping agent for many years. Although not clinically approved for human application, Bo or its main metabolite 5β-androst-1-en-17β-ol-3-one (BoM) were detected in several doping control samples (1). The possibility of endogenous Bo production in human beings has been discussed. This is a challenging issue for doping control laboratories as Bo belongs to the list of prohibited substances of the World Anti-Doping Agency (WADA) and is usually found at low concentrations. Anti-doping laboratories must have available methods capable of detecting synthetic steroids at concentrations below 10 ng mL-1 and, at the same time, be able to quantify endogenous steroids with accuracy. When atypical concentrations of endogenous steroids are measured, a complementary analysis using gas chromatography combustion isotope ratio mass spectrometry (GC-C-IRMS) must be carried out and besides the unequivocal identification of the steroids must be carried out by GC-MS (2). In this communication we describe a new method base on a first separation of unacetyl-steroids by RPLC followed by on-line coupled liquid chromatography-gas chromatography (LC-GC) using the Through Oven Transfer Adsorption Desorption (TOTAD) interface and mass spectrometer (MS) detector to analyze Bo and BoM. By this means, all the analyte eluted in the LC stage is automatically transferred to the GC, avoiding the contamination associated with manipulation of the sample. A fraction collector permits the two fractions eluted in LC to be stored and sent individually to the GC by means of the TOTAD interface, once the sample is derivatized, is completely automatic. The detection limits attained, measured in GC-MS in SCAN mode, is below 0.5 ng/mL for Bo and below 0.05 ng/mL for BoM. The presented LC-GC-MS method could be used to confirm the identity of the compounds analyzed by LC-GC-C-IRMS (3).

References:

(1)          Piper, T et al. Drug. Test. Analysis, 2 (2010) 217

(2)          WADA Technical Document – TD2014IRMS

(3)          Toledano, R.M. et al. J. Chromatogr. A, 1370 (2014) 171

Toxicological Center, University of Antwerp, Universiteitsplein 1, 2610 Antwerp, Belgium, This email address is being protected from spambots. You need JavaScript enabled to view it.

Ethylphenidate is a new psychoactive substance (NPS) that recently appeared on the drug market as an alternative for methylphenidate, and it was already found responsible of a human fatality. The identification of (human) metabolites is a necessary task, for example to confirm the consumption of a particular drug when the drug is metabolized and, sometimes, not observed in urine. The aim of this study was to investigate the in vitro Phase-I and Phase-II metabolites of ethylphenidate using human liver microsomes (HLMs) and human liver cytosol (HLCYT). The metabolites formed were identified by liquid chromatography (LC) coupled to tandem mass spectrometry (MS/MS) using a quadrupole/time-of-flight (QTOF) as analyser. Six Phase-I, but no Phase-II metabolites were detected. Ethylphenidate underwent hydroxylation forming two primary mono-hydroxylated metabolites and, subsequently, dehydration and ring opening with an additional hydroxylation, forming secondary metabolites. In addition, ritalinic acid and methylphenidate were formed, but through non-enzymatic hydrolysis and subsequent methylation, and, therefore, they are not considered metabolites of ethylphenidate. The involvement of different human cytochrome P450 (CYP) enzymes in the formation of ethylphenidate metabolites showed that rCYP2C19 was the most active enzyme being involved in the formation of all the six ethylphenidate metabolites detected, although also other rCYPs (rCYP1A2, rCYP2B6, rCYPC9, rCYP2D6, and rCYP3A4, but not rCYP2E1) participated in the metabolism of ethylphenidate.

All metabolites identified in the present study are candidate specific biomarkers for monitoring studies, including toxicological screening. However, the presence of methylphenidate and ritalinic acid cannot be solely associated to ethylphenidate intake because methylphenidate is a drug itself and ritanilic acid can be formed also from methylphenidate by hydrolysis.

Acknowledgements

A-TEAM (#316665) and SEWPROF (#317205) Marie Curie Training Networks are acknowledged. N.N. and A.L.N.v.N. acknowledge the University of Antwerp and FWO Flanders, respectively, for their postdoctoral fellowships.

Faculty of Chemistry, Maria Curie-Skłodowska Univ, Pl. M. Curie Skłodowska 3, 20-031 Lublin, POLAND, This email address is being protected from spambots. You need JavaScript enabled to view it.

Determination of free drug fraction in human serum is a very important issue in pharmacokinetics research. Drugs can reversible binding with a plasma proteins (especially HAS and AGP) formation complexes. Only unbounded part of the drug can caused a therapeutic effect. There are a lot of factors influencing on the bound a given drugs with a serum proteins – so the researches are very needed. There are a lot of method for determination of free drug fraction. Some problem with a method is isolation of proteins from the analytical system. The operations can change a free drug fraction value. Liquid chromatography methods are vide used for determination of the free drug fraction in systems without proteins. We proposed use Micellar Liquid Chromatography (MLC) for determination of the free drug fraction in two cases:

Fist as a method for determination of the free form of the drug in deprived of proteins system, second as direct sample injection analysis (DSI). MLC is a chromatographic method friendly to environment due to small consuming organic solvents. Surfactants used in MLC are biodegradable. Replacement RP-LC chromatography MLC is advantageous for natural environment . In the second hand, due to ability to solubilisation of the proteins and theirs complexes in micelles MLC can be used as DSI of samples containing proteins. In presented investigations results of MLC-DSI analysis were compared with a result obtained by adsorption method. Two adsorption systems, with a two adsorbents can be tested, and free drug (ampicillin) fraction can be determined by spectrophometric, RP-HPLC and MLC method. MLC systems, with a different stationary phase (RP-8e and RP-CN) and surfactants (SDS and CTAB) were tested for DSI analysis. The system consists of CTAB and RP-8e column which proved to be the most useful in the evaluation of the concentration of the free ampicillin fraction in HSA solutions (the obtained values were the similar to the values obtained by the use of adsorption method: RP-HPLC and spectrophotometric analyses).

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

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

3 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.

The present research is focused on the development of a new versatile multidimensional MDGC preparative system coupled, whenever required by the complexity of the sample, to an LC pre-separation step operated in normal phase. The system demonstrated to be capable of collecting sample components in a wide range of concentrations, in a short time period, while maintaining a high degree purity for the collected components. When necessary, the LC dimension allows the injection of higher sample amounts and the transfer of simplified sub-samples to the MDGC system equipped with a programmed temperature vaporizer injector (PTV). Furthermore, thanks to the LC pre-separation step it was possible to reduce the matrix effect due to the transfer of selected LC chromatographic bands, diluted in a pure organic solvent (the LC mobile phase), characterized by low viscosity, simultaneously reducing the possibility to introduce in the GC injector and columns non-volatile components. The system versatility enhances the collection of pure components from complex samples in a green manner thanks to the reduced number of runs required. In fact, depending on the complexity, as well as to the relative concentration of the components to be collected in the whole sample,the system can be operated in LC-GC-GC or in GC-GC-GC mode. Finally, by using a commercially available collection station with a 10-position carousel it was possible to exploit a multi collection step per run.