Urine Testing for Designer Steroids by Liquid Chromatography with Androgen Bioassay Detection and Electrospray Quadrupole Time-of-Flight Mass Spectrometry Identification

Abstract

New anabolic steroids show up occasionally in sports doping and in veterinary control. The discovery of these designer steroids is facilitated by findings of illicit preparations, thus allowing bioactivity testing, structure elucidation using NMR and mass spectrometry, and final incorporation in urine testing. However, as long as these preparations remain undiscovered, new designer steroids are not screened for in routine sports doping or veterinary control urine tests since the established GC/MS and LC/MS/MS methods are set up for the monitoring of a few selected ions or MS/MS transitions of known substances only. In this study, the feasibility of androgen bioactivity testing and mass spectrometric identification is being investigated for trace analysis of designer steroids in urine. Following enzymatic deconjugation and a generic solid-phase extraction, the samples are analyzed by gradient LC with effluent splitting toward two identical 96-well fraction collectors. One well plate is used for androgen bioactivity detection using a novel robust yeast reporter gene bioassay yielding a biogram featuring a 20-s time resolution. The bioactive wells direct the identification efforts to the corresponding well numbers in the duplicate plate. These are subjected to high-resolution LC using a short column packed with 1.7-microm C18 material and coupled with electrospray quadrupole time-of-flight mass spectrometry (LC/QTOFMS) with accurate mass measurement. Element compositions are calculated and used to interrogate electronic substance databases. The feasibility of this approach for doping control is demonstrated via the screening of human urine samples spiked with the designer anabolic steroid tetrahydrogestrinone. Application of the proposed methodology, complementary to the established targeted urine screening for known anabolics, will increase the chance of finding unknown emerging designer steroids, rather then being solely dependent on findings of the illicit preparations themselves.

Challenges in detecting substances for equine anti‐doping

“…Moreover, detection of unknown designer steroids is difficult in biological samples either using specific analysis or mass spectral library search, as both approaches would require establishing the analytical characteristics with suitable reference materials. [52][53][54] The detection strategies have been thoroughly reviewed elsewhere. [55] It is not uncommon that doping agents used in human sports would likewise be abused in equine sports.…”

Colorimetric detection and chromatographic analyses of designer drugs in biological materials: a comprehensive review

“…The drug groups and their structures are shown in Figs. 1,2,3,4,5,6,7,8,9,10,11. The PEA derivatives usually imitate amphetamines in their effects.…”

“…The majority of the drugs have hallucinogenic effects. Recently, steroid designer drugs have been detected in urine samples obtained from athletes [8][9][10][11][12][13].…”

Bioassay based screening of steroid derivatives in animal feed and supplements

“…Supplemented minimal medium (MM/L) consisted of yeast nitrogen base without ammonium sulphate or amino acids (1.7 g L −1 ), dextrose (20 g L −1 ), ammonium sulphate (5 g L −1 ) and was supplemented with l-leucine (6 mg L −1 ). The yeast androgen and estrogen bioassays were performed as described previously [3,14]. In short, 10 mL MM/L was inoculated with a single colony of the recombinant yeast and grown overnight at 30 • C in an orbital shaking incubator at 125 rpm.…”

“…Hormone abuse or incidents may be discovered by residue analysis in matrices such as urine, hair and feed. For screening animal feed and supplements reporter gene bioassays have proven their added value in detecting known and unknown steroidal compounds [3][4][5]. Oral administration of natural steroids via feed or supplements results in low bioavailability due to poor intestinal absorption and extensive hepatic first-pass metabolism.…”