Development Of New Analytical Methods For Steroid Profiling For Doping Purposes
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Date
2018-07
Authors
Abdul Manaf, Normaliza
Journal Title
Journal ISSN
Volume Title
Publisher
Universiti Sains Malaysia
Abstract
The steroid profile compounds namely testosterone (T), epitestosterone (E),
androsterone (A), etiocholanolone (Etio), 5alpha-androstane-3alpha,17beta-diol
(5αAdiol), 5beta-androstane-3alpha,17beta-diol (5βAdiol) and the ratio of T to E
(T/E)) has been determined in urine of male volunteers who consumed Tongkat Ali
(TA) supplement using the solid phase extraction (SPE) and liquid-liquid extraction
(LLE) method. From 47 samples, 11 samples violated the World Anti-Doping
Agency (WADA) guidelines and confirmation by gas chromatography combustion
isotope ratio mass spectrometry (GC-C-IRMS) showed 5 samples were consistent
with the exogenous origin (Δδ13C > 3‰). Two microextraction methods, i.e., vortexassisted
liquid-liquid microextraction (VALLME) and micro-solid phase extraction
(μ-SPE) followed by LC-MS/MS were developed. Hypersil Gold C18 column (50
mm × 2.1 mm, 1.9 μm) with gradient elution resulted in baseline separation for
compounds in about 8 min. The monitored ions are m/z 289.4 > 97.3 for T and E,
m/z 273.4 > 255.3 for A and Etio and m/z 275.4 > 257.3 for 5αAdiol and 5βAdiol
using electron spray ionization in the positive polarity mode. The optimum extraction
conditions for 5 mL sample using the VALLME were: extraction solvent, 1-
pentanol; volume of extractant, 150 μL; vortex time, 40 s; centrifuge speed and time,
1000 rpm for 1 min with no salt addition needed for the extraction. The optimum
extraction conditions for 3 mL sample using the μ-SPE were: extraction solvent,
acetonitrile; volume of extractant, 300 μL; extraction time, 30 min and desorption
time, 20 min for the extraction. The VALLME-LC-MS/MS and μ-SPE-LC-MS/MS
methods meet WADA requirements in terms of sensitivity, limit of detection,
reproducibility, linearity and robustness. Furthermore, these alternative techniques
were simple, rapid and environmentally friendly as markedly reduced amounts of
solvents were involved.
Description
Keywords
Development of new analytical methods , steroid profiling for doping purposes