Steroid detection and identification remain key issues in toxicology, drug testing, medical diagnostics, food safety control, and doping control. In this study, we evaluate the capabilities and usefulness of analyzing non‐hydrolyzed sulfated steroids with gas chromatography−mass spectrometry (GC–MS) instead of the conventionally applied liquid chromatography−mass spectrometry (LC–MS) approach. Sulfates of 31 steroids were synthesized and their MS and chromatographic behavior studied by chemical ionization−GC−triple quadrupole MS (CI−GC‐TQMS) and low energy−electron ionization−GC−quadrupole time‐of‐flight−MS (LE−EI−GC−QTOF−MS). The collected data shows that the sulfate group is cleaved off in the injection port of the GC–MS, forming two isomers. In CI, the dominant species (ie, [MH – H2SO4]+ or [MH – H4S2O8]+ for bis‐sulfates) is very abundant due to the limited amount of fragmentation, making it an ideal precursor ion for MS/MS. In LE−EI, [M – H2SO4].+ and/or [M – H2SO4 – CH3].+ are the dominant species in most cases. Based on the common GC–MS behavior of non‐hydrolyzed sulfated steroids, two applications were evaluated and compared with the conventionally applied LC–MS approach; (a) discovery of (new) sulfated steroid metabolites of mesterolone and (b) expanding anabolic androgenic steroid abuse detection windows. GC–MS and LC–MS analysis of non‐hydrolyzed sulfated steroids offered comparable sensitivities, superseding these of GC–MS after hydrolysis. For non‐hydrolyzed sulfated steroids, GC–MS offers a higher structural elucidating power and a more straightforward inclusion in screening methods than LC–MS.

Sulfated metabolites have been shown to have potential as long‐term markers of anabolic–androgenic steroid (AAS) abuse. In 2019, the compatibility of gas chromatography–mass spectrometry (GC–MS) with non‐hydrolysed sulfated steroids was demonstrated, and this approach allowed the incorporation of these compounds in a broad GC–MS initial testing procedure at a later stage. However, research is needed to identify which are beneficial. In this study, a search for new long‐term metabolites of two popular AAS, metenolone and drostanolone, was undertaken through two excretion studies each. The excretion samples were analysed using GC–chemical ionization–triple quadrupole MS (GC–CI–MS/MS) after the application of three separate sample preparation methodologies (i.e. hydrolysis with Escherichia coli–derived β‐glucuronidase, Helix pomatia–derived β‐glucuronidase/arylsulfatase and non‐hydrolysed sulfated steroids). For metenolone, a non‐hydrolysed sulfated metabolite, 1β‐methyl‐5α‐androstan‐17‐one‐3ζ‐sulfate, was documented for the first time to provide the longest detection time of up to 17 days. This metabolite increased the detection time by nearly a factor of 2 in comparison with the currently monitored markers for metenolone in a routine doping control initial testing procedure. In the second excretion study, it prolonged the detection window by 25%. In the case of drostanolone, the non‐hydrolysed sulfated metabolite with the longest detection time was the sulfated analogue of the main drostanolone metabolite (3α‐hydroxy‐2α‐methyl‐5α‐androstan‐17‐one) with a detection time of up to 24 days. However, the currently monitored main drostanolone metabolite in routine doping control, after hydrolysis of the glucuronide with E.coli, remained superior in detection time (i.e. up to 29 days).

No abstract

Quantification of IGF-I is relevant in both doping control as a biomarker of growth hormone (GH) misuse in sports, and in the clinical field for longitudinal follow-up of patients with disorders related to the GH axis. Currently, better standardization of IGF-I measurements using mass spectrometry is in our best interest as it would enable long-term monitoring of an athletes' IGF-I levels by its addition to the Athlete Biological Passport (ABP). Here, a simplified and rapid top-down LC-HRMS method for quantification of IGF-I in human serum is presented. A ten-minute precipitation-based offline sample preparation is combined with online sample clean-up and separation on a conventional LC, resulting in a total runtime of nine minutes in between injections. The method was validated in the relevant range of 50-1000 ng/mL for the following parameters: linearity, precision, bias, Limit Of Quantification (LOQ), carry-over, selectivity, recovery and ion suppression. As proof of concept, the presented LC-HRMS assay was compared with results from a previous inter-laboratory study on intact IGF-I quantification using four human GH administration samples. It was additionally compared with the IDS-iSYS immunoassay using 47 athlete serum samples, showing good overall agreement with a slight positive bias of 24.2 ng/mL for the LC-HRMS assay at a mean sample concentration of 234 ng/mL. Also, a discrepancy between commercially available IGF-I reference material for the calibration of quantitative assays is discussed. This is of importance if LC-MS assays for IGF-I are to be harmonized. Recently, an inter-laboratory study compared these different approaches, finding no significant difference in quantification [38]. Bottom-up approaches require more laborious sample preparation which induces additional analytical variation through its trypsin digestion step [39]. As harmonization of IGF-I quantification would be to the benefit of both doping control laboratories and clinical laboratories, such variation should be avoided as much as possible by targeting the intact protein in a top-down approach.

Human insulin and its synthetic analogs are considered as life‐saving drugs for people suffering from diabetes mellitus. Next to the therapeutic use, scientific and non‐scientific literature (e.g. bodybuilding forums; antidoping intelligence and investigation reports) indicate that these prohibited substances are used as performance enhancing agents. In the present report, the development and validation of a sensitive analytical strategy is described for the urinary detection of three rapid‐acting insulin analogs (Lispro, Aspart, Glulisine). The method is based on sample purification by the combination of ultrafiltration and immunoaffinity purification and subsequent analysis by nano‐flow liquid chromatography coupled to high resolution mass spectrometry. Next to the results on different validation parameters (LOD: 10 pg/mL; recovery: 25–48%; matrix effect: −3‐(−8) %), data on urinary elimination times, which were obtained in the frame of an administration study with the participation of healthy volunteers, are presented. The determined detection windows (~9 hours) are expected to help to evaluate current routine analytical methods and aim to aid doping authorities to set appropriate target windows for efficient testing.