been demonstrated.8 Recently, Labrie et al.9 reported the results
of their studies regarding the potential anabolic/androgenic
activity of THG by identifying its effect on the expression of the
mouse genome (near 30 000 genes) and with respect to the effect
of dihydrotestosterone (DHT), the most potent natural androgen.
Both steroids modulated the same genes in a similar manner.
However, although under in vivo conditions THG possesses 20%
of the potency of DHT in stimulating prostate, seminal vesicle,
and certain muscles in mouse, THG was found more potent than
DHT in binding to the AR. Therefore, there is a high risk for
undesirable effects on the health status of individuals consuming
this type of hormone.
of the analysis/sample, ease of use, selectivity, and detectability
to analyze small organic molecules (i.e. refs 14 and 15). Thus,
immunochemical analytical methods have proved to be appropriate
tools for the screening other anabolic steroids used as doping
agents or growth promoters in farm animals.16-18 Moreover, in
this case availability of antibodies may help to identify other
metabolites or bioconjugates structurally related with THG as well
as to complete the THG ADMET (adsorption, distribution,
metabolism, excretion, and toxicity) profile. Attending to these
facts, this paper presents for the first time the preparation of
antibodies and the necessary immunoreagents for THG determi-
nation. These immunoreagents can be the base for further
development of bioanalytical methods based on different protocols
(i.e., immunoaffinity extraction procedures, immunoassay, western-
blot, etc.) and sensing principles (i.e., optical, electrochemical,
or piezoelectric immunosensors) as well as other biochemical
investigations. Here we present the characterization of the
antibodies through the development of an ELISA protocol for the
detection of THG. The assay has proven to be useful to analyze
this substance in human urine samples.
Detection of drug use is not a trivial analytical task. Before
routine implementation, there are many questions to be answered
such as the type of sample to be collected, identification of the
proportion between the parent compound and the metabolites
present in a particular type of sample, selection of an efficient
extraction procedure, and a selective and sensitive detection
method, etc. For drugs that have routine medical applications,
information on the pharmacokinetic/pharmacodynamics is usually
available. However, athletes often use nonapproved anabolic
steroids or those approved only for veterinary applications. Since
metabolism is different in animals and in man, human excretion
needs to be determined. Regarding THG, no legitimate in vivo
human excretion studies identifying urinary markers of this doping
agent have been reported. The most reliable information available
belongs to in vitro systems using human hepatocytes. According
to these studies based on HPLC/MS/MS and NMR data, an in
vitro metabolic pathway leading to the addition of a hydroxyl group
followed by the â-glucuronic acid at C-18 has been proposed as
the major metabolic route. Second, the formation of a glucoronide
conjugate of an oxidative product with a hydroxyl group probably
at C-16 has been suggested.10 The information reported from these
in vitro experiments have provided the basis for further identifica-
tion of THG human urinary markers. Meanwhile, analytical
chromatographic methodologies have been established, aimed at
detecting THG related substances,10-12 to perform these metabolic,
biochemical, and activity studies. In an effort to discover new
designer steroids early, Nielen et al.13 have recently reported the
combination of a novel yeast reporter gene androgen bioassay
with HPLC coupled to time-of flight mass spectrometry (LC/
QTOFMS) which allows accurate mass measurements of the
bioassay positive results. Alternatively, the high-sample throughput
capability of the immunochemical methods could respond to the
demands of the control for the illegal use of this drug by elite
amateur and professional athletes. It has been often demonstrated
that immunoassays can provide the necessary reliability, low cost
EXPERIMENTAL SECTION
General Methods and Instruments. Thin layer chromatog-
raphy was performed on 0.25 mm precoated silica gel 60 F254
aluminum sheets (Merck, Gibbstown, NJ), and the separations
of the different compounds synthesized were done by column
chromatography with silica 60 A C.C. 35-70 µm sodium dodecyl
sulfate. 1H and 13C NMR spectra were obtained with a Varian
Inova-500 (Varian Inc., Palo Alto, CA) spectrometer (500 MHz for
1H and 125 MHz for 13C). Infrared spectra were measured on a
Bowmen MB120 FT-IR spectrophotometer (Hartmann & Braun,
Que´bec, Canada). HPLC-UV analysis were performed using a
Merck Hitachi L-7100 pump provided with a diode array L-7455
detector, a L-7200 autosampler, and a D7000 interface (Merck,
Darmstadt, Germany). The chromatograms were processed with
the HSM software (Merck, Darmstadt, Germany). The column
used was Lichrospher 100 RP-18 125 × 4 (5 µm; Merck,
Darmstadt, Germany), and the analyses were performed on
isocratic mode using acetonitrile (ACN):H2O 6:4 as mobile phase
at a flow rate of 1.0 mL min-1. The reactions were monitored at
three wavelengths: 345, 310, and 254 nm. Preparative HPLC was
performed using a Waters Prep LC4000 pump (Millipore Corp.,
Milford, MA) and a Waters Prepack 1000 pressure module with
a flow distributor where 2% of the sample goes to the Merck-
Hitachi L-4000 detector (Merck, Darmstadt, Germany). The
column was a Perkin-Elmer Preparative C18 Flow, and the mobile
phase was passed at a flow rate of 12 mL min-1 with the following
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Baeza, F.; Marco, M.-P. In Emerging Organic Pollutants in Waste Waters
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Analytical Chemistry, Vol. 79, No. 10, May 15, 2007 3735