Development and application of novel methods for the detection of anabolic androgenic steroids in hair and other matrices using liquid chromatography tandem mass spectrometry

Deshmukh, Nawed Inayat Khan (2013) Development and application of novel methods for the detection of anabolic androgenic steroids in hair and other matrices using liquid chromatography tandem mass spectrometry. (PhD thesis), Kingston University, uk.bl.ethos.587388.

Abstract

Owing to an increase in the number of doping cases with performance enhanc-ing drugs (PEDs), mainly anabolic androgenic steroids (AASs), and the limitations of urinalysis and self-reports, there is an ever increasing need to develop new methods for detecting doping. This thesis reports the development and application of a series of nov¬el analytical methods for the detection of frequently used AASs in hair and other matri¬ces using liquid-chromatography tandem mass-spectrometry (LC-MS/MS). Assays capable of detecting 0.5 pg/mg stanozolol and 3.0 pg/mg nandralane (with 20 mg hair) were developed. Hair samples from 180 subjects (108 males, 72 fe-males) were screened using ELISA, which revealed 16 subjects to be positive for stanozolol and 3 for nandrolone. LC-MS/MS analysis confirmed that only 11 subjects were positive for stanozolol (5.0 pg/mg to 86.3 pg/mg) and just 1 for nandralane (14.0 pg/mg), thus showing the inaccuracy of using ELISA screening alone. The analytical fmdings were successfully employed to verify self-reported drug use data. An assay capable of detecting 0.1 pg/mg testosterone (T) and 0.25 pg/mg epitestosterone (E) (with 50 mg hair) was developed. Seventy-five hair samples were collected from healthy volunteers (49 males, 26 females), with the natural levels of T 0.7-11.81 pg/mg and 0.33-6.05 pg/mg and the natural levels ofE 0.63-8.27 pg/mg and 0.52-3.88 pg/mg, in males and in females respectively. This thesis reports for the first time the TÆ ratio in hair, 0.5-3.37 in males and 0.56-1.81 in females. Assays capable of detecting 0.125 pg/mg stanozolol/0.25 pg/mg 3'-hydroxystano-zolol (with 50 mg rat hair) and 0.063 ng/mL stanozolol/0.125 ng/mL 3'-hydroxystanozolol (with 100 uL of rat urine or serum) were developed. Diclofenac was found to significantly reduce the urinary excretion of 3'-hydroxystanozolol, but did not influence the concentration of both compounds in hair. This is the first in vivo study to report this effect. Assays capable of detecting 0.25 pg/mL stanozolol and 0.5 pg/mL of 3'-hydroxystanozolol in 5 mL aqueous matrices were developed in order to investigate en-vironmental contamination. Three out of six samples from the River Danube, collected from December '09 to November '10, were found to contain stanozolol, (upto 1.82 pg/mL). In contrast, stanozolol was detected only once (1.19 pg/mL) in tap water from Budapest city.

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