Urine testing for designer steroids by liquid chromatography with androgen bioassay detection and electrospray quadrupole time-of-flight mass spectrometry identification.

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.

Multi residue screening of intact testosterone esters and boldenone undecylenate in bovine hair using liquid chromatography electrospray tandem mass spectrometry.

The abuse of esters of natural androgenic steroids in cattle fattening and sports is hard to control via routine urine testing. The esters are rapidly hydrolysed in vivo into substances which are also endogenously present in urine. In veterinary control strange findings of 17beta-testosterone and 17alpha-testosterone in urine are often ignored because of the lack of statistically sound reference data of naturally occurring levels. An interesting alternative for inconclusive urine analyses in veterinary control can be provided by the analysis of the administered steroids themselves, i.e. the analysis of intact steroid esters in hair. Unfortunately, the analysis of intact steroid esters is complicated not only by the vulnerability of the esters which precludes alkaline hydrolysis of the hair, but also by the wide polarity range of short and long-chain esters yielding very poor recoveries for either the one or the other. In this study, a multi-steroid esters LC/MS/MS screening method is presented for trace analysis of the synthetic intact esters of 17beta-testosterone and the undecylenate ester of 17beta-boldenone in bovine hair. The method, requiring only 200 mg of pulverised hair, features a mild digestion procedure using tris(2-carboxyethyl)phosphine hydrochloride (TCEP) and the use of four deuterium-labelled steroid esters as internal standards covering the wide polarity range of the analytes. In spiked hair samples for most of the analytes the limit of detection and the accuracy using isotope dilution were 2-5 ng/g and 97-105%, respectively. The applicability was demonstrated using hair samples from a controlled experiment in which six bovines were injected intramuscularly with two different doses of two commercial mixtures of testosterone esters, and with two different doses of boldenone undecylenate. Depending on the dose all administered testosterone- and boldenone esters were found to be incorporated in bovine hair following a single intramuscular injection, except testosterone propionate which dose might have been too low.

Bioassay-directed identification of estrogen residues in urine by liquid chromatography electrospray quadrupole time-of-flight mass spectrometry.

A new approach to the search for residues of known and unknown estrogens in calf urine is presented. Following enzymatic deconjugation and solid-phase extraction, a minor part of the samples is screened for estrogen activity using a recently developed rapid reporter gene bioassay. The remainder of the bioactive extracts is analyzed by gradient liquid chromatography (LC) with, in parallel, bioactivity and mass spectrometric detection via effluent splitting toward a 96-well fraction collector and an electrospray quadrupole time-of-flight mass spectrometer (QTOFMS). The LC fractions in the 96-well plate are used for the detection of estrogen activity using the bioassay. The biogram obtained features a 20-s time resolution, and the suspect well numbers can be easily correlated with the LC/QTOFMS retention time. The mass spectral data from the thus assigned relevant parts of the chromatograms are background subtracted, followed by accurate mass measurement, element composition calculation, and identification. The method allows estrogen activity detection and identification of (un)known estrogens in urine at the 1-2 ng/L level, in compliance with current residue analysis performance for hormone abuse in cattle. The applicability of this LC/bioassay/QTOFMS approach for the identification of estrogens in real-life samples is demonstrated by the analysis of several calf urine samples, and preliminary data from a pig feed sample.