6890n series

For enantioselective analysis, an Agilent gas chromatograph (model 6890N series) was used coupled to a mass spectrometer detector (quadrupole model Agilent series 5973 inert) using an enantioselective column with a stationary phase (2,3-diethyl-6-tert-butyldimethylsilyl-β-cyclodextrin). The chromatographic run was performed with a temperature ramp of 2 °C/min from 50 °C (mainatined for 2 min) to 220 °C (mainatined for 2 min).

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.

Metabolomic analysis was performed on an Agilent 7683B series injector (Agilent, Santa Clara, CA) coupled to an Agilent 6890 N series gas chromatograph system and a 5975 mass selective detector (MSD) (Agilent, Santa Clara, CA). Aglient DB-5MS column (0.25 μm, 0.25 mm × 30 m, Agilent Technologies, Inc., Santa Clara, CA) was used. The column temperature was 70 °C for the first 4 min and then increased at 5 °C/min to 310 °C for 15 min.The injection temperature was set as 300 °C, and the injection volume was 1 μL with a 10:1 split ratio. Helium (99.9995%, China) was applied as a carrier gas. The column flow was 1.2 mL/min, and the column was equipped with a linear velocity control model. Prior to the instrumental analysis, the mass spectrometer was tuned using perfluorotributylamine (PFTBA) to obtain optimum performance. A simultaneous full scan-selected ion monitoring mode (Scan-SIM) was used to acquire the data. The mass spectra scanning scope was set to 33–600 m/z in the full scan mode and 90 chromatographic peaks of 25 groups were set in the selected ion monitoring. The scan speed is 2.59 scan s-1 and the solvent cut time is 5.0 min. The temperatures of the interface and the ion source were adjusted to 280 and 230 °C, respectively. The detector voltage was maintained at 1.2 kV, and the electron impact (EI) model was selected to achieve ionization of the metabolites at 70 eV.