Dissertation committee:
Μ. Κουππάρης Καθηγ.(επιβλέπων), Α. Καλοκαιρινός Καθηγ., Τ. Αττά-Πολίτου Αναπλ. Καθηγ. , Κ. Ευσταθίου Καθηγ., Α. Οικονόμου Επικ. Καθηγ., Ν. Θωμαΐδης Επικ. Καθηγ., E. Μπακέας Επικ. Καθηγ.
Summary:
Critical requirement in doping control laboratories is the identification of a
large number of prohibited substances with different properties, in low
concentrations, found in different biological fluids, such as urine. Nowadays,
most doping control laboratories follow different sample preparation and the
respective analytical procedures for this purpose. The development of a unified
screening method is necessary for saving cost, time and human resources.
The present work, carried out in the framework of WADA funded research project,
investigates the development of a unified screening method for the
identification of prohibited substances, in athlete urine samples, through the
use of GC/TOFMS technology. The screening method includes a common sample
preparation and the respective analytical protocol. The first involves the
hydrolysis, extraction and derivatization steps.
The experimental protocol for the development of a unified derivatization
method was focused on the formation of TMS-derivatives. The optimization of the
proposed derivatization process was followed. The optimized two-step procedure,
with a total duration of 20 min, allowed for the detection of 190 prohibited
substances in a concentration equal to the Minimum Required Performance Level
(MRPL).
The experimental protocol for the development of a unified extraction procedure
includes four experiment series. At first, the solid phase (SPE) and
liquid-liquid extraction (LLE) protocols were studied in unhydrolysed urine
samples. The combination of SPE and LLE resulted in a cleaner background and
satisfying results in regard to the detection of the compounds of interest. The
aforementioned protocols together with those of exclusive LLE extraction were
tested on hydrolyzed urine. As a result of these tests, 9 protocols giving high
recovery and low variance were applied for the extraction of the total number
of compounds in urine samples at MRPL. Eventually, LLE with diethylether as
extraction solvent was selected.
The application of the unified method includes the analysis by GC/TOFMS, of
urine samples that were prepared by the unified sample preparation procedure.
For this reason, the retention times with the main diagnostic ions for the
compounds of interest, were firstly defined. Macros were modified in the
instrument software for the automation of data processing. 169 prohibited
substances were identified with S/N greater than 3 and excellent mass accuracy
out of 183 substances that were studied in urine samples at MRPL. Part of those
substances that were not identified with GC/TOFMS can be identified with
LC/TOFMS.
The unified screening method allows for the identification of large number of
substances in athletes’ urine samples with good performance due to its high
resolution and mass accuracy in full scan mode.
Keywords:
Screening method, Trimethylsilyl-derivatives, Liquid-Liquid extraction, Solid Phase Extraction, GC/TOFMS
