Energy-Resolved Fragmentation Aiding the Structure Elucidation of Steroid Biomarkers

Abstract

The identification and confirmation of steroid sulfate metabolites in biological samples are essential to various fields, including anti-doping analysis and clinical sciences. Ultra-high-performance liquid chromatography with tandem mass spectrometry (UHPLC-MS/MS) is the leading method for the detection of intact steroid conjugates in biofluids, but because of the inherent complexity of biological samples and the low concentration of many targets of interest, metabolite identification based solely on mass spectrometry remains a major challenge. The confirmation of new metabolites typically depends on a comparison with synthetically derived reference materials that encompass a range of possible conjugation sites and stereochemistries. Herein, energy-resolved collision-induced dissociation (CID) is used as part of UHPLC-HRMS/MS analysis to distinguish between regio- and stereo-isomeric steroid sulfate compounds. This wholly MS-based approach was employed to guide the synthesis of reference materials to unambiguously confirm the identity of an equine steroid sulfate biomarker of testosterone propionate administration.

Evaluation of alternative gas chromatographic and mass spectrometric behaviour of trimethylsilyl‐derivatives of non‐hydrolysed sulfated anabolic steroids

“…The last two fractions were collected, dried, residues dissolved in 500 μL MeOH and kept at −20°C until use. The synthesis procedure has been evaluated in several previous publications, 16–20 and the SPE‐fraction containing the synthesised sulfated reference material was analysed using LC–MS by monitoring of the of [M‐H] − → 97 ([HSO 4 ] − ) To additionally determine the success of the reaction, 50 μL of both solutions were dried, derivatised and analysed using GC‐EI‐QTOF‐MS.…”

“…The last two fractions were collected, dried, residues dissolved in 500 μL MeOH and kept at À20 C until use. The synthesis procedure has been evaluated in several previous publications, [16][17][18][19][20] and the SPE-fraction containing the synthesised sulfated reference material was analysed using LC-MS by monitoring of the of…”

New long‐standing metabolites of 17α‐methyltestosterone are detected in HepG2 cell in vitro metabolic model and in human urine

“…AASs are the most misused class of substances according to the WADA global testing figures, 1,2 and often the parent compound is not excreted at all in post‐administration urine samples, or it is excreted for a limited period of time 3–7 . AASs are metabolized extensively to both phases I and II metabolites in humans, and despite the in‐depth studies on their biotransformation routes performed in previous decades, the need for more efficient markers of abuse justifies ongoing studies using state‐of‐the‐art instrumentation 8–11 . It is important to note that for the efficient monitoring of AASs misuse in the frame of sports drug testing, the most abundant metabolites are not necessarily the best markers of AASs administration, but instead, the best markers are those which provide the longest period of detectability (long‐term metabolites).…”

Very Low-Pressure CID Experiments: High Energy Transfer and Fragmentation Pattern at the Single Collision Regime

“…Increasing the collision energy increases the internal energy of the colliding ions, and that in turn increases the degree of fragmentation, changes the ratio of various fragment ion abundances, and may also change fragmentation routes. Varying and optimizing the collision energy has various practical applications, such as determination of binding energies [ 8 , 9 ], differentiation of isomers [ 10 , 11 , 12 ], characterization of oligonucleotides [ 13 ] and small molecules [ 14 , 15 , 16 , 17 ], and various proteomic applications [ 18 , 19 , 20 ] including quantitation [ 21 ].…”