Session 10

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.