Detection of mono‐hydroxylated metabolites of stanozolol by HPLC‐ESI (+) MS/MS in Indian sports persons

Abstract

The abuse of stanozolol is quite widespread in Indian sport. Its analysis is challenging and this has led to the development of new methods to improve its detection. A method was developed and validated for the detection of the three main monohydroxylated metabolites of stanozolol. The excretion profile of these metabolites was studied in four healthy male volunteers. The excretion study samples, after a single oral dose of drug, showed that 3'-OH-stanozolol was excreted at the highest concentration, followed by 16beta-OH stanozolol, with 4beta-OH stanozolol as the least excreted. Ninety-eight old doping samples with adverse analytical findings for 3'-OH-stanozolol were reanalysed using this method. This showed 3'-OH-stanozolol and 16beta-OH stanozolol in all the 98 samples whereas 4beta-OH-stanozolol was identified in 90 samples. The percentage of positive identifications of stanozolol in Indian sportspeople has increased markedly in the last five years, from 31.9% in 2004 to 81.8% in 2009; however, this may be due to the more effective detection of stanozolol metabolites. It can thus be concluded that the marked increase in percent positive of stanozolol in Indian sportspersons in 2009 may be due to the improved detection by a more effective LCMS/MS method.

Simultaneous identification of prednisolone and its ten metabolites in human urine by high performance liquid chromatography‐tandem mass spectrometry

“…The sample preparation procedure involves enzymatic hydrolysis and liquid‐liquid extraction . Based on the specific gravity (SG) of the urine samples, 2 ml (SG ≥ 1.010) or 4 ml (SG < 1.010) of urine sample aliquot was taken; 500 ng/ml of internal standard (17‐α methyl testosterone) was added to each sample aliquot.…”

“…The sample preparation procedure involves enzymatic hydrolysis and liquid-liquid extraction. [27] Based on the specific gravity (SG) of the urine samples, 2 ml (SG ≥ 1.010) or 4 ml (SG < 1.010) of urine sample aliquot was taken; 500 ng/ml of internal standard (17-a methyl testosterone) was added to each sample aliquot. Hydrolysis was done at pH 7.0 using 0.2 M phosphate buffer by b -glucuronidase (E.coli) enzyme at 60 C for 1 h. The pH was adjusted to 9-10 with 7% K 2 CO 3 and liquid-liquid extraction was performed using 5 ml TBME.…”

Detection of stanozolol O‐ and N‐sulfate metabolites and their evaluation as additional markers in doping control

“…[9][10][11][12][13] The first metabolic products described were the monohydroxylated derivatives 3'-hydroxystanozolol (3STAN), 4β-hydroxy-stanozolol (4STAN) and 16β-hydroxy-stanozolol (16STAN) (Figure 1, 2-4) as well as corresponding analogues epimerized in C-17 ( Figure 1, 5). [9][10][11][12][13] The first metabolic products described were the monohydroxylated derivatives 3'-hydroxystanozolol (3STAN), 4β-hydroxy-stanozolol (4STAN) and 16β-hydroxy-stanozolol (16STAN) (Figure 1, 2-4) as well as corresponding analogues epimerized in C-17 ( Figure 1, 5).…”

Expanding analytical possibilities concerning the detection of stanozolol misuse by means of high resolution/high accuracy mass spectrometric detection of stanozolol glucuronides in human sports drug testing

“…[1] Among these AAS, stanozolol (Figure 1, 1) in particular proved challenging due to its peculiar physico-chemical properties (arising from the steroidal A-ring bearing a condensed pyrazole moiety), complicating the formerly exclusively employed detection assays based on gas chromatography-mass spectrometry (GC-MS). [6][7][8][9][10] The molecular features of stanozolol and its metabolites demanding a sophisticated derivatization and tweaked chromatography with GC-MS-based methodologies proved extraordinarily advantageous for liquid chromatography-electrospray ionization-(tandem) mass spectrometry [LC-MS(/MS)]based approaches, [5,11,12] resulting in continuously decreasing limits of detection (LODs) and detection windows with expanding spectra of target analytes, the latest contribution to which being 3'-OH-stanozolol glucuronide (Figure 1, 3) analyzed at 25-50 pg /ml in human urine. [6][7][8][9][10] The molecular features of stanozolol and its metabolites demanding a sophisticated derivatization and tweaked chromatography with GC-MS-based methodologies proved extraordinarily advantageous for liquid chromatography-electrospray ionization-(tandem) mass spectrometry [LC-MS(/MS)]based approaches, [5,11,12] resulting in continuously decreasing limits of detection (LODs) and detection windows with expanding spectra of target analytes, the latest contribution to which being 3'-OH-stanozolol glucuronide (Figure 1, 3) analyzed at 25-50 pg /ml in human urine.…”

“…In 1986, the analysis of stanozolol abuse by means of a major urinary metabolite referred to as 3'-OH-stanozolol (Figure 1, 2) as accomplished by Schänzer and Donike, [2] initiating in-depth investigations into the metabolic fate of this frequently observed AAS [3][4][5] and options to improve detection times after cessation of its use by elite athletes. [6][7][8][9][10] The molecular features of stanozolol and its metabolites demanding a sophisticated derivatization and tweaked chromatography with GC-MS-based methodologies proved extraordinarily advantageous for liquid chromatography-electrospray ionization-(tandem) mass spectrometry [LC-MS(/MS)]based approaches, [5,11,12] resulting in continuously decreasing limits of detection (LODs) and detection windows with expanding spectra of target analytes, the latest contribution to which being 3'-OH-stanozolol glucuronide (Figure 1, 3) analyzed at 25-50 pg /ml in human urine. [13] In the present study, the use of high resolution/high accuracy mass spectrometry for the detection of 3'-OHstanozolol glucuronide is outlined, and complementary information on N-conjugated glucuronide metabolites of stanozolol and 17-epistanozolol and the use of these in routine doping controls is provided.…”