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Realised by ALMS™
developer of the AIDS-HIV Reference project
Abstract No.: TuP-090
Session: Forensics and Security Aspects
Presentation date: Tue, Aug 29, 2006
Presentation time: 14:30 – 16:00

Determination of 3-keto-4,9,11-triene Steroids, β-2 Agonists, Oxandrolone and Oralturinabol in Human Urine by High Performance Liquid Chromatography – Ion Trap Mass Spectrometry

Marina A. Dikunets, Svetlana A. Appolonova, Grigory M. Rodchenkov

1 Antidoping Centre, Moscow, Russian Federation

Correspondence address: Marina A. Dikunets, Antidoping Centre, Elizavetinskiy per.10, Moscow, 105005 Russian Federation.

Keywords: Chromatography, Liquid -, High Pressure (HPLC); Ionization, Electrospray; Mass Spectrometry, Ion Trap Quadrupole; Steroids.

Novel aspect: New Approach for simultaneous determination of β-2 agonists, 3-keto-4,9,11-triene steroids and anabolic androgenic steroids by HPLC-ESI-MS.


Β-2 agonists such as fenoterol, salmeterol, formoterol are commonly used for the treatment of pulmonary diseases such as asthma bronchial. 3-Keto-4,9,11-triene steroids (gestrinone, tetrahydrogestrinone, trenbolone) have antiprogestagenic and anti-estrogenic properties. Oralturinabol and oxandrolone are anabolic androgenic steroids. These substances are considered as doping and included in the List of prohibited substances issued by the World Anti–Doping Agency (WADA).
The high-performance liquid chromatography coupled to atmospheric pressure electrospray ionization – ion trap mass spectrometry (HPLC–ESI–MSn) method was applied for the determination of above substances in human urine. A positive ion mode was used in the detection. In the LC-ESI-MS and LC-ESI-MS-MS experiments the detection was based on selected ion monitoring (SIM) and isolation and further fragmentation of the [M+H]+ ions, respectively.
Sample pretreatment was performed by enzymatic hydrolysis and liquid-liquid extraction (mixture of diethyl ether with toluene). The organic fraction was evaporated to dryness. The residue was dissolved in 50 µl of methanol and 5 µl of the solution was injected into the HPLC system. Separation was achieved at 30°C using a 100x2.1 mm I.D., 5 µm Restek Ultra C18 column connected to a guard column 4x12.5 mm. The mobile phase was 0.2 mM ammonium acetate with 0.05% formic acid (A) – acetonitrile (B) at flow-rate of 0.2 ml/min. The gradient mode was following: 0 min – 5% B; 10 min – 30% B; 15 min – 60% B; 25 min – 85% B. The limits of quantification were about 1 ng/ml for fenoterol, salmeterol, formoterol, gestrinone, tetrahydrogestrinone, trenbolone, oralturinabol and oxandrolone in human urine. The HPLC-ESI-MS Ion Trap procedure showed high sensitivity, good linearity and reproducibility.