Detecting the AAS have been able to reduce

Detecting misuse of AAS in athletes is based on finding
the presence of specific steroids in urinary samples by detecting the urinary phase-I
or phase-II long-term exogenous AAS metabolites (table 1). The detection methods available for
the AAS have been able to reduce its use from 3,297 being identified positive
for misuse to 1,728 in 2015 by the laboratory statistical analysis by
WADA-laboratories (WADA et al., 2015). The most common direct detection
methods are performed using urine specimen as matrix for doping control purpose,
samples are collected from the athlete and partitioned into two aliquots (A-
and B-sample), which are separately sealed. The first screening analysis on the
A-sample, which covers a wide variety of steroid metabolites that are either in
unconjugated form or glucuronides. This is followed by the analysis of a second
aliquot of the same specimen in case of a suspicious result in screening
(Flenker U. et al., 2008). Only after confirmation of the screening result the
responsible Anti-Doping Organisation and the WADA are informed about the
adverse analytical finding; it allows the prolongation of the detection window
and an increased retrospectivity for AAS. For sample preparation aliquots are deconjugated
by ?-glucuronidase enzyme and extraction of the deconjugated steroids from the
matrix and concentration of the analyses is performed by liquid–liquid
extraction or solid phase extraction followed by mass spectrometric detection
either by liquid chromatography tandem mass spectrometry (LC-MS/MS) (Ahrens BD
et al., 2011) or gas chromatography (tandem) mass spectrometry (GC-MS(/MS)). GC
is used in hyphenation with combustion isotope ratio mass spectrometry (GC?C?IRMS) a
highly specialized instrumental confirmatory technique,
measuring the carbon isotope
ratio (?13C) of urinary steroids and confirming
their synthetic origin based on the variation in the 13C/12C
ratio of the testosterone metabolites will depend on 13C
depletion (indicator of AAS misuse) of the pharmaceutical preparation
administered, as well as the athlete’s baseline values from the ABP and
individual metabolism. The indirect detection in AAS doping is based on
endocrinology feedback mechanism, which is detecting the ratio of testosterone
and epitestosterone (T/E), that is determined from the ratios of the corrected
chromatographic peak areas as peak heights, and if ratio is greater than 4
there is suspicion of doping.