Summary:
In the current study, new qualitative and quantitative methods were developed
for the doping control screening analysis, confirmation, and/or quantitative
determination of prohibited substances using LC/TOF-MS technology. The first
part of this work presents the use of LC/TOF-MS for the multiple detection of a
great number of prohibited substances in athletes urine. In the second part,
the same technology was applied for the quantification and identification of
threshold substances in horse urine..
Unification of screening protocols for a wide range of doping agents has become
an important issue for doping control laboratories. In the present study, a
high throughput screening method was developed for the multiple detection of
266 small molecule analytes from all categories of prohibited substances
included in the WADA List. The proposed methodology is based on a single-step
liquid-liquid extraction of hydrolyzed urine and the use of a rapid-resolution
LC/TOF-MS system acquiring continuous full scan spectral data. The sample
preparation procedure used in this study was applied in the OACA laboratory for
the routine screening analysis of anabolic agents. Our approach was to study
the efficiency of this procedure for other classes of substances. Validation
parameters consisted of identification capability, limit of detection,
specificity, ion suppression, extraction recovery, repeatability and mass
accuracy. Detection criteria were established on the basis of retention time
reproducibility and mass accuracy. The suitability of the methodology for
doping control was demonstrated with positive urine samples. The preventive
role of the method was proved by the case where full scan acquisition with
accurate mass measurement allowed the retrospective reprocessing of acquired
data from past doping control samples for the detection of a designer drug, the
stimulant 4-methyl-2-hexanamine.
In the second part of this work, LC/MS methods were developed for the
quantification of four threshold substances (hydrocortisone, theobromine,
salicylic acid and 3-methoxytyramine) in horse urine. The main purpose was the
application of minimal sample preparation and direct injection analysis of
diluted horse urine samples into the LC/MS system. The study of matrix effect
and the selection of an appropriate methodology proved to be important issues
during method development for the accurate quantification of the analytes.
Identification (confirmation) of the analytes was achieved in the same
analytical protocol with the quantification and in some cases in the same
analytical run. Validation results for all methods proved their suitability for
the doping control analysis of all analytes in horse urine. The developed
methods were applied to the analysis of real positive horse urine samples.
Keywords:
Doping control, Liquid chromatography/mass spectrometry, Time of flight mass spectrometry, Athletes screening analysis, Matrix effect
