Use of LC-MS/MS for the Open Detection of Steroid Metabolites Conjugated with Glucuronic Acid

In humans, conjugation with glucuronic acid is the most important phase II metabolic reaction of steroidal compounds. Glucuronoconjugated metabolites have been conventionally studied by using β-glucuronidase enzymes to release the phase I metabolites. It is well-known that hydrolysis with β-glucuronidase presents some limitations that may result in the underestimation of some conjugates. The aim of the present work was to develop and to evaluate liquid chromatography-tandem mass spectrometry (LC-MS/MS) scan methods for the open detection of steroid glucuronides in urine samples. The mass spectrometric behavior of thirteen representative steroid glucuronides, used as model compounds, was studied. Characteristic ionization and collision induced dissociation behaviors were observed depending on the steroid glucuronide structure. Neutral loss (NL of 176, 194, 211, and 229 Da) and precursor ion (PI of <i>m</i>/<i>z</i> 141, 159, and 177, in positive mode and <i>m</i>/<i>z</i> 75, 85, and 113, in negative mode) scan methods were evaluated. The NL scan method was chosen for the open detection of glucuronoconjugated steroids due to its sensitivity and the structural information provided by this method. The application of the NL scan method to urine samples collected after testosterone (T) undecanoate administration revealed the presence of two T metabolites which remain conjugated as glucuronides after an enzymatic hydrolysis of the urine. 3α,6β-Dihydroxy-5α-androstan-17-one (6β-hydroxyandrosterone) glucuronide and 3α,6β-dihydroxy-5β-androstan-17-one (6β-hydroxyetiocholanolone) glucuronide were established as the structures for these metabolites, by comparing the structure of the steroids released after chemical hydrolysis with reference materials. An increase of 50–300-fold of these metabolites after oral administration of T undecanoate was observed, proving that their determination can be useful in the doping control field. Moreover, these results exemplify that significant information might be missed, unless direct methods for the determination of steroid glucuronides are employed

Untargeted Metabolomics in Doping Control: Detection of New Markers of Testosterone Misuse by Ultrahigh Performance Liquid Chromatography Coupled to High-Resolution Mass Spectrometry

The use of untargeted metabolomics for the discovery of markers is a promising and virtually unexplored tool in the doping control field. Hybrid quadrupole time-of-flight (QTOF) and hybrid quadrupole Orbitrap (Q Exactive) mass spectrometers, coupled to ultrahigh pressure liquid chromatography, are excellent tools for this purpose. In the present work, QTOF and Q Exactive have been used to look for markers for testosterone cypionate misuse by means of untargeted metabolomics. Two different groups of urine samples were analyzed, collected before and after the intramuscular administration of testosterone cypionate. In order to avoid analyte losses in the sample treatment, samples were just 2-fold diluted with water and directly injected into the chromatographic system. Samples were analyzed in both positive and negative ionization modes. Data from both systems were treated under untargeted metabolomic strategies using XCMS application and multivariate analysis. Results from the two mass spectrometers differed in the number of detected features, but both led to the same potential marker for the particular testosterone ester misuse. The in-depth study of the MS and MS/MS behavior of this marker allowed for the establishment of 1-cyclopentenoylglycine as a feasible structure. The putative structure was confirmed by comparison with synthesized material. This potential marker seems to come from the metabolism of the cypionic acid release after hydrolysis of the administered ester. Its suitability for doping control has been evaluated