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

• Main Author: Deshmukh, Nawed Inayat Khan
• Format: Dissertation
• Language: English
• Published: Kingston University 2013
• Subjects: Biological sciences; Chemistry; Pharmacy; Pre-clinical and human biological sciences

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.