5973 series

Frozen samples of semen were thawed at room temperature, and subsequently, the vials were immersed in a water bath onto a magnetic stirrer hotplate at 60 °C overnight.
After this incubation, a Carboxen^®/Polydimethylsiloxane (CAR/PDMS) fiber (57318, Supelco) was exposed to headspace for 15 min, as reported in [32 (link)]. GC-MS analysis of extracted volatiles was performed using GC (6890N series Agilent Technologies) coupled to MS (5973 series Agilent Technologies) equipped with a ZB-624 capillary column (Phenomenex); the injector temperature was set at 250 °C to allow thermal desorption of VOCs. The carrier gas was high-purity helium with a flow rate of 1 mL min⁻¹. The MS analyses were carried out in full-scan mode with a scan range of 30–500 amu at 3.2 scans/s. Chromatograms were analyzed by Enhanced Data Analysis (MSD Chemstation E.02.02, Agilent Technologies, Santa Clara, CA, USA), and the identification of the volatile compounds was achieved by comparing mass spectra with those of the data system library (NIST14, p > 60%) and confirmed by the injection of external standards corresponding to most recurrent compounds. To quantify the identified VOCs, a semiquantitative method based on the internal standard (I.S.) 1,4-Dichlorobenzene-D4 (EPA-8260C) was followed. A frequency analysis was performed to identify the most frequently present VOCs.

Calibration standards and brominated samples were analyzed using Agilent Technologies GC 6980 series fitted with a mass spectrometry detector 5973 Series and HP-Innowax capillary column (30 m × 0.25 mm, i.d., 0.25 mm film thickness (Agilent Technologies, Palo Alto, CA, USA). An aliquot of 2 µL was first injected in a split-less mode (split flow of 60 mL/min) at 250 °C using an auto-sampler (Agilent Technologies Series 7683, Palo Alto, CA, USA) with a purge activation time of 1 min. Helium was selected as a carrier gas set at a flow rate of 1.6 mL/min. The column was fixed at 65 °C for 1 min and gradually programmed at 15 °C/min to 170 °C, followed by 5 °C/min to 200 °C and 40 °C/min to 250 °C before held for 15 min at 250 °C. The selected ion monitoring (SIM) mode was used, and the m/z 149 for 2-bromopropenamide and 154 for 2-bromo(13C3)propenamide ions were monitored throughout the instrumentation process.