Complementary information concerning the suspected interindividual transmission of GW1516, a substance prohibited in sport, through intimate contact: a case report

Acetonitrile (ACN), methanol and formic acid were obtained from VWR chemicals (Langenfeld, Germany). Ammonium acetate and acetic acid were obtained from Merck (Darmstadt, Germany). Ultrapure water was received from a Barnstead GenPure xCAD Plus from Thermo Scientific (Bremen, Germany). The Rapid Stain Identification (RSID™)-semen field test was obtained from Independent Forensics (Lombard, IL). The seminal fluid samples were collected as part of routine andrological examinations at Muenster University Hospital (Germany) and consent was obtained for further research purposes.

To test for the presence of semenogelin, a lateral flow immunochromatographic analysis was performed as an initial-testing procedure as described by Breuer et al., 2021 [13]. In brief, 20 µL of urine was diluted with 80 µL RSID™ Universal Buffer and transferred on the lateral flow immune strip. Positive and negative controls were prepared and analyzed as recommended. Ten minutes after application, the result (presence or absence of semen) was photographically documented.

The LC–MS/MS analysis was performed as described by Breuer et al., 2021 [13]. Briefly, after preparation of the urine samples by solid-phase extraction, tryptic digestion was performed. The analysis for the presence of three specific peptides of semenogelin I was conducted using a Vanquish™ UHPLC system coupled to a Thermo Scientific Orbitrap Exploris™ 480 mass spectrometer (Thermo Scientific, Bremen, Germany). Characteristics of peptides of semenogelin and the internal standard (ISTD) used for the LC–MS/MS assay are listed in Table 1.

To 100 µL of seminal fluid, 5 μL of an ISTD working solution was added and mixed with 100 µL acetic acid (1%). To precipitate proteins, 800 µL of ice-cold ACN was added, and the sample was vortexed for 20 s and cooled for 20 min at 4°C. Following centrifugation (15 min, 14,000 × g), the pellet was discarded and the supernatant was evaporated to dryness in a vacuum centrifuge. The dried sample was reconstituted in 100 μL of H₂O/ACN (80/20, v/v).

LC–MS/MS analysis of seminal fluid was conducted using an Aquity I-Class ultra-performance liquid chromatograph (UPLC) coupled to a Xevo Triple Quadrupole-XS mass spectrometer (TQ-MS), both from Waters (Eschborn, Germany). Seminal fluid samples prepared for analysis were injected onto a Poroshell C-8 analytical column (50 × 3.0 mm, 2.7 μm particle size; Agilent, Waldbronn, Germany) employing the eluents A (0.1% formic acid in water) and B (0.1% formic acid in ACN) at a flow rate of 0.4 mL/min. The method started at 5% B, increasing to 15% B from 1 to 2 min, followed by an increase to 55% B from 2 to 11 min. B was then increased to 100% from 11 to 12 min, followed by equilibration at 1% B from 12 to 15 min. Before the next injection, the needle was washed for 1 min while B was increased from 1% to 5%. The compounds were introduced into the mass spectrometer by electrospray ionization and were detected by time-based multiple-reaction monitoring experiments. The data were processed using TargetLynx™ (Waters, Eschborn, Germany) after analysis.

A LC–HRMS method was developed for the analysis of GW1516 and its metabolites. The LC–HRMS system used was a Vanquish HPLC system coupled to a Thermo Scientific Orbitrap Exploris™ 480, both from Thermo Scientific (Bremen, Germany). For chromatography, a Poroshell 120 EC-C18 analytical column (50 × 3.0 mm, 2.7 μm particle size; Agilent, Waldbronn, Germany) was used, connected to an EC 4/3 Nucleoshell RP 18 Plus guard column (4 × 3 mm, 5 μm particle size) from Macherey–Nagel (Düren, Germany). The gradient with a run time of 15 min and a flow rate of 0.4 mL/min was performed using water containing 0.1% acetic acid as solvent A and ACN containing 0.1% acetic acid as solvent B. The method was started with 0% B, increasing to 90% B in 11 min. Subsequently, 100% B was held for 1 min followed by re-equilibration at 0% B for 3 min. The injection volume was 5 µL. Measurements were conducted in positive ionization mode with an ionization voltage of 3 kV and a transfer tube temperature of 320 °C. A resolution of 60,000 full width at half maximum (FWHM) was selected for the full-scan and 45,000 FWHM for HRMS/MS experiments, and the full-scan was performed in a range of m/z 100–800. The normalized collision energy (NCE) and precursor ions are listed in Table 2. For product ion scan experiments, the isolation window of the quadrupole was set to 1 m/z. Nitrogen was generated by the CMC nitrogen generator (Eschborn, Germany) and used as collision gas. The Thermo Scientific Orbitrap Exploris™ 480 was calibrated regularly with the manufacturer’s calibration solution.