Quantification Of Endogenous Anabolic Androgenic Steroids Using Gas Chromatography Tandem Mass Spectrometry; An Application Of Steroid Profile From Eurycoma Longifolia Administration
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Date
2011
Authors
Abushareeda, Wadha Masoud
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Abstract
A method was developed using gas chromatography tandem mass spectrometry (GC/MS/MS) to quantify the levels of endogenous androgenic anabolic steroids (EAAS) including testosterone, dehydroepiandrosterone, 4-andro -stenedione, 5-androstenediol, dihydrotestosterone and other related metabolites like androsterone, etiocholanolone, 5-α-androstandiol and 5-β-androstandiol in urine samples. The steroids were extracted using SPE method followed by LLE and analyzed by GC/MS/MS. Multiple Reaction Monitoring (MRM) was adopted for the analysis, using two MRM transitions as the qualifier ions and one MRM transition as the quantifier ion for each analyte. The linearity, extraction recovery, LOD, intra- and inter–day precision and accuracy as well as robustness of the method were validated. The correlation coefficients (r2) obtained was higher than or equal to 0.993 for all the compounds. The recoveries achieved using the method were higher than 70% for all analytes The limits of detection that was obtained using the method described in the present study ranged from 0.1 to 0.2 ng/mL. The limits of quantification ranged from to 0.5 to 1 ng/mL. The results showed good precision; where CV < 8% and accuracy ≥ 80% for all compounds in all assayed levels. The validation results showed that the analytical method used in this study to quantify the investigated anabolic steroids in urine has complied with the criteria of the validation for quantitative methods according to WADA ISL and NATA Technical Note.EAAS is a group of natural compounds that are chemically similar to testosterone and share the same androgenic anabolic action. Administration of these substances for enhancement of athletic performance is forbidden in sport. Abuse of these steroids is one of the most serious violations in sport. Eurycoma longifolia is an herbal plant native to South East Asian countries which is claimed to boost the testosterone levels. Accordingly, it is used among the athletes and bodybuilders to enhance the muscle mass, strength, and the overall performance. In this study, 27 volunteers were recruited to consume Eurycoma longifolia over a period of 15 days and the urine sample of each subject was collected every five days. Quantification of EAAS after administration of Eurycoma longifolia was carried out using GC-MS/MS method. Results of the administration study showed a significant increase during the first five days in the urinary levels of the testosterone and related steroid precursors and metabolites. Although there were no significant differences in the levels of EAAS between the athletes and non-athletes group as well as between treated and placebo group, there were clearly significant differences in the levels as a function of time.
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Keywords
Anabolic steroids , Doping in sports