Krzysztof Gorynski (Presenter)
Nicolaus Copernicus University Collegium Medicum
Bio: Dr. Krzysztof Goryñski (MALE) holds a M.Sc. (2008; with excellence), and a Ph.D. (2014; with hons) from Collegium Medicum, Nicolaus Copernicus University, Toruñ, Poland. He is assistant professor (adiunct) in Department of Pharmacodynamics and Molecular Pharmacology. He spent in total almost 3 years as a visiting scientist in Prof. Janusz Pawliszyn laboratory in University of Waterloo, Canada. The visit gave him the opportunity to work on the application of the solid-phase microextraction (SPME) method for clinical analysis in collaboration with Toronto General Hospital. He also actively participated in the project “Evaluation of Solid Phase Microextraction for improved Multi-Residue Extraction and Analysis of Prohibited Substances by LC-MS/MS” supported by the World Anti-doping Agency (WADA). His research works were presented on several (18 oral and 30 posters)international conferences.
Authorship: Krzysztof Gorynski(1*), Lukasz Sobczak(1), Michael Pasek(1), Paulina Gorynska(1), Janusz Pawliszyn(2), Barbara Bojko (1,2)
(1) Department of Pharmacodynamics and Molecular Pharmacology, Faculty of Pharmacy, Collegium Medicum in Bydgoszcz Nicolaus Copernicus University in Toruñ, Poland; (2) Department of Chemistry, University of Waterloo, Ontario, Canada
One of the tasks of the doping control laboratories is applying reliable techniques that are capable of multi-compound analysis at least on cut-off concentration levels. Today, the determination of substances in complex biological matrices cannot be performed without proper sample preparation, even when using highly powerful and efficient analytical instrumentation, such as LC-MS. Solid Phase Microextraction (SPME) is a sampling and sample preparation technique that has indicated its great potential for analysis of prohibited substances from complex matrices. Ongoing studies expend utilization of the technology for saliva analysis as an alternative specimen to urine and blood during doping control testing. Thus, in this talk we will describe recent developments in SPME relevant to the potential applications toward doping control of urine, plasma, blood or alternative matrices.
Despite the variety of analytical protocols available for anti-doping screening, there is still a need for development of new technologies that can provide improved sample preparation from various matrices for simultaneous multi-compound analysis. Since there are a number of compounds that can be potentially used as performance enhancers by sportsmen, the chosen method should be able to screen as many drugs of interest as possible in one single analysis, without sacrificing sensitivity or time length of analysis. Solid-phase microextraction (SPME) is a well-established sample preparation technique introduced to analytical community two decades ago and have recently made significant advances when applied to variety of exo- and endogenous compounds including drugs and metabolites analysis. SPME is solvent free technique which provides very efficient sample clean-up due to the use of very small volume of extraction phase what limits the possible interferences and eliminates matrix effect in LC-MS analysis. Moreover it is applicable to complex heterogenous samples with no need of sample pretreatment and can be applied to a small sample volumes without significant loss of sensitivity. Recent preliminary studies have shown its great potential in the analysis of doping substances in urine, blood and plasma, as regular matrices applied for doping control, as well as saliva, as alternative fluids (1,2).
The Solid Phase Microextraction was developed and applied as sample preparation method before LC-MS analysis. Protocol was optimized in terms of extraction pH, ionic strength of the sample, washing solution, extraction and desorption times. We determined also analytical figures of merit for all target compounds for which acceptable sensitivity and throughput were achieved. We determined limit of detection, lower limit of quantification, upper limit of quantification, linear dynamic range, relative standard deviations through the dynamic range, absolute and relative recoveries and matrix effect. The validation was completed according FDA procedure and WADA requirements. SPME was coupled to: high sensitive LC-MS platform, triple quadrupole Shimadzu LC-MS 8060 and high-resolution instrument Thermo Exactive Orbitrap.
The applicability of SPME for analysis of prohibited substances in urine, plasma, blood and most recently alternative matrix, saliva, will be discussed. Up-to-date results show limits of detection at pg/mL levels, thus showing potential of the approach to be used as fast and sensitive tool for screening of prohibited substances in alternative matrices e.g. oral fluid/saliva. The comparison of different sample collection technique (salivette vs. spitting) and two different sample preparation method (SPME vs. PP) in combination with high resolution mass spectrometer provide extend information about quality and quantity of metabolites presence depends which configuration was applied. Optimized protocol of SPME (in terms preconditioning solvent, pH of extraction, desorption solvent) for determination of 46 compounds from saliva matrix will be presented with figure of merits from validation steps. The results also demonstrate that SPME is suitable for simultaneous multi-compound bioanalysis of analytes with a wide range of polarities using single protocol allowing analysis time to be briefer than traditional sampling procedures. Results from comparison to commercially available saliva test against narcotics vs. SPME will be presented. Also data obtained from matrix effect studies (PP vs. SPME) will be discussed.
Conclusions & Discussion
The proposed high-throughput method requires very little handling of samples for determination of compounds from many classes of prohibited doping substances representing a wide range of physicochemical properties. The proposed SPME method for analysis of doping substances has numerous advantages when it is compared with currently used approaches. It offers fully automated and high-throughput monitoring of substances simultaneously, allowing analysis time to be shorter than traditional sampling procedures. The proposed protocol is simple and does not require any sample pretreatment, provides excellent clean-up, while preventing from occurring matrix effects, one of the main drawbacks of standard procedures. The SPME in 96-thin-film format has been shown to be a powerful tool that makes sample preparation no longer a bottleneck of doping analysis; this can be accomplished due to the automated sampling methodology, which can decrease response time during particular sport events. In turn, development of analytical protocol available for anti-doping control of saliva offers sampling in a non-invasive way, with no adulteration during sample collection, and which supervision during collection would not compromise the privacy of the tested athletes.
References & Acknowledgements:
 E. Boyacý, K. Gorynski, A. Rodriguez-Lafuente, B. Bojko, J. Pawliszyn, Anal. Chim. Acta 809 (2014) 69-81
 N. Reyes-Garcés, B. Bojko, J. Pawliszyn, J. Chromatogr. A, 1374 (2014) 40-49
IP Royalty: no
|Planning to mention or discuss specific products or technology of the company(ies) listed above:||