SOFT 2004 Meeting Abstracts - Society of Forensic Toxicologists

F3
ANALYSIS OF URINARY STEROID SULFATE METABOLITES USING ION-PAIRED
EXTRACTION
'12
Ad am Cawley " Rymantas Kazlauskas
l , Graham Trout l , Adrian George 2
1. Australian Sports Drug Testing Laboratory, National Measurement Institute, Sydney, Australia, 2.
School ofChemistry, University of Sydney, Sydney, Australia
Urinary steroid metabolites may be excreted in their unchanged (free) form, and/or more likely, as
glucuronide or sulfate conjugates. Routinely, doping control laboratories accredited by the International
Olympic Committee (JOC) and the World Anti-Doping Agency (WADA) screen urine samples collected
from athletes for the presence of free and glucuronide conjugated steroids by Gas Chromatography-Mass
Spectrometry (GC-MS) following enzymatic hydrolysis and solid phase extraction. There is a clear need,
however, for laboratories to analyse a number of steroids excreted primarily as their sulfate conjugates in
order to further metabolic research and develop confirmation methods that are more specific to particular
steroid administrations. Published methods utilising a range of chemical hydrolysis techniques to cleave the
sulfate moiety from steroid metabolites and make them amenable to GC-MS analysis have been
problematic to implement due to urinary matrix effects. To circumvent this problem, ion-pairing extraction
with (-)-N,N-dimethylephedrinium bromide under basic conditions was optimised to obtain a purified
sulfate fraction. Cleavage of the sulfate moiety was efficiently achieved by chemical hydrolysis under mild
conditions using methanolic-HCI derived from trimethylchlorosilane (TMCS). The free steroids were
extracted into hexane at pH 9.8 before reaction with N-methyl-N-(trimethylsilyl)trifluoroacetamide
(MSTFA) to form TMS enol ether derivatives that were subsequently analysed by GC-MS. This method
provided linear recoveries (R 2 2:0.9993, 100 to 5000 ng/mL) of greater than 90% for androsterone (5aandrostane-3a-oI-17-one)
extracted as the sulfate conjugate. The potential of direct urinary sulfate
metabolite analysis using Liquid Chromatography-Mass Spectrometry (LC-MS) is also discussed as a
means to alleviate the need for chemical hydrolysis. While these methods are directed at steroid detection
for IOCfW ADA laboratories they may be applied in a broader toxicological sense for the analysis of
sulfated metabolites of other drugs of abuse. Novel analysis methods such as these may also allow more
detailed metabolic studies to be carried out to determine new markers of specific drug abuse, thus
demonstrating their potential as a valuable confirmation technique. Incorporated into the GC-MS method
validation was an evaluation of a reasonable estimate of measurement uncertainty that is presented as an
example to emphasise the importance ofISO 17025 compliance for forensic toxicology laboratories.
Keywords: urinary sulfate metabolites, ion-paired extraction, measurement uncertainty
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