1290 infinity

The total lipid fraction was extracted with a 1:1 chloroform–methanol mixture from the sperm cells pellet, as previously described [38 (link)]. After resuspension in 200 μL of chloroform, the sterol fraction was isolated from lyophilized extracts by solid-phase extraction (SPE) on a 1-mL silica column, eluted with acetone and lyophilized in a Vacufuge^®-Concentrator plus. The eluate was dissolved in methanol and 20 μL were analyzed by reverse-phase (RP) liquid-chromatography coupled to high-resolution mass spectrometry (LC-MS). A Poroshell 120 EC-C18 column (4.6 mm × 150 mm, 2.7-μm particle size) from Phenomenex (Torrance, CA, USA) was equilibrated and eluted at 40 °C with an acetonitrile/methanol solution (65:35 v/v), at a flow rate 0.3 mL/min using an Agilent (Santa Clara, CA, USA) 1290 Infinity Ultra-High Performance Liquid Chromatography (UPLC) system, equipped with a 920 autosampler and connected to a Waters (Milford, MA, USA) Xevo-G2S Q-TOF mass spectrometer, as previously described [13 (link)].

Soluble proteins from POSs resuspended in DPBS were analyzed using HPLC coupled to an ultraviolet (UV) detector. HPLC-UV analyses were performed with Agilent Technologies 1290 Infinity (Santa Clara, CA, USA) using a Waters BioResolve C4 column (150 × 2.1 mm, 2.6 μm) (Milford, MA, USA). Mobile phases were composed of 0.1% (v/v) TFA in water (A) and 70% acetonitrile with 30% isopropyl alcohol (B), with an elution gradient to equilibrate the system as follows: 0 to 2 min, 32%; 2 to 22 min, 32 to 85%; 22 to 24 min, hold at 85%; 24 to 25, 85 to 32%; and 5.5 min. The temperature of the column was set at 70 °C, with a flow rate of 0.4 mL/min. UV detection was performed at 214 and 280 nm using a diode array detector (DAD). The data were analyzed with Agilent MassHunter (B.07.00, Agilent, Santa Clara, CA, USA).