Agilent 1290 infinity 2 uplc

For intra-testis testosterone quantification, testis samples were homogenized twice using the Precellys 24 tissue homogenizer (Bertin Instruments; Rockville, MD, USA) (4°C, 3×, 30 s at 6500 rpm, cycle break 30 s) in chloroform-isopropanol (1 mL, 50/50%; v/v) containing ISTD. Combined supernatants were centrifuged (10 min, RT, 16,000 × g) and evaporated using a Genevac EZ-2 evaporator (Stepbios, Muttenz, Switzerland) (3 hr, 35°C). Samples were reconstituted in methanol (50 µL, 10 min, RT, 1300 rpm) and sonicated (10 min, RT). Reconstituted samples were centrifuged (10 min, RT, 16,000 × g), and supernatants were transferred to LC-MS vials. Testosterone content was analyzed by ultra-performance liquid chromatography-MS/MS (UPLC-MS/MS) using an Agilent 1290 Infinity II UPLC coupled to an Agilent 6495 triple quadrupole mass spectrometer equipped with a jet-stream electrospray ionization interface (Agilent Technologies). Analyte separation was achieved using a reverse-phase column (1.7 µm, 2.1 mm × 150 mm; Acquity UPLC BEH C18; Waters). Data acquisition and quantitative analysis were performed by MassHunter (Version B.10.0. Build 10.0.27, Agilent Technologies).

Soil and plant samples were extracted using solid-phase extraction (SPE) following the method previously described by Chen et al. [29 (link)]. Briefly, three extractions were performed on plant and soil samples using acetonitrile/water (v/v = 9:1) and acetonitrile. The extracts were then concentrated with nitrogen and underwent extraction clean-up using Oasis WAX SPE tubes as detailed in the Supplementary Materials. PFAS were quantified in negative electrospray ionization (ESI) mode using an Agilent 1290 infinity II UPLC (Agilent Technologies, Santa Clara, CA, USA) combined with an API 5500 triple-quadrupole mass spectrometer (AB SCIEX Inc., Framingham, MA, USA). Separation of the target PFAS was performed using the Waters acquity BEH C18 column (1.7 μm, 2.1 mm × 100 mm). Detailed descriptions of the instrumental analyses and mass spectral parameters for the target compounds are given in Tables S4 and S5.

Pooled and dried samples were resuspended in 20 μl 2% (vol/vol) acetonitrile for fractionation. Basic reversed phase fractionation was performed on a quaternary Agilent 1290 Infinity II UPLC system equipped with a Kinetex Evo-C₁₈ column (150 by 2.1 mm, 2.6 μm, 100 Å, Phenomenex) that was operated at 40°C. Solvent A consisted of high-performance liquid chromatography (HPLC)-grade H₂O, solvent B consisted of 100% acetonitrile, and solvent C consisted of 25 mM ammonium bicarbonate. Fractionation was carried out at a constant flow rate of 100 μl/minute using a linear gradient from 2 to 25% (vol/vol) acetonitrile within 50 min, followed by column washing and equilibration. Over the whole gradient, solvent C was kept constant at 10% (vol/vol). In total, 32 fractions with 200 μl per fraction were collected in conical 96-well plates (Greiner). The organic solvent was removed in a vacuum concentrator (default settings for organic solvents) for 1 h and fractions were concatenated into 8 final samples as described before (64 (link)). Peptides were acidified with formic acid (Thermo Fisher Scientific) to a final concentration of 1% (vol/vol), desalted using OASIS HLB 96-well cartridges (Waters), dried down at room temperature, and resuspended in 30 μl 2% (vol/vol) acetonitrile, 0.1% (vol/vol) trifluoroacetic acid (TFA) prior to mass spectrometry (MS) analysis.