Infinity 2 uhplc

For the biotransformation, 22% NADES (BSorbW or BEtGly), 0.9 mM piperine, 590 µL LOX_SA (32 U/mL), 1.5 mM linoleic acid and 100 µL of 20 mM sodium phosphate buffer (pH 7) were added to a 4 mL glass vial. The total volume of the reaction was 1 mL.
As a control, experimental setups with inactive enzyme and with Britton–Robinson buffer (pH adjusted to the pH of the respective NADES) instead of an NADES were used. The mixture was shaken at 200 rpm and 22 °C for 24 h and the reaction was stopped with 1 mL of 99% methanol (HPLC grade) and filtered. As an internal standard, 1 µL of 160 mM ferulic acid ethyl ester was added. The measurements were performed on an Infinity II UHPLC (Agilent Technologies, Santa Clara, CA, USA) using water + 0.1% formic acid as solvent A and acetonitrile as solvent B. An amount of 1 µL of sample was injected into a Poroshell 120 EC-C18 (2.7 µm, 2.1 × 100 mm). The gradient used was 95% A, 0–2 min; 95 to 85%, 2–7 min; 85 to 50%, 7–13 min; 50 to 20%, 13–14 min; 20%, 14–16 min; 20 to 95%, 16–18 min; and 95% A, 18–20 min. The flow rate was set to 0.5 mL/min and the column oven temperature to 25 °C. The chromatograms were evaluated at 354 nm using a DAD detector.

Reduced and total 3SH were analyzed by SIDA-UPLC-MS/MS following a previously established method (Roland et al., 2016 (link)) based upon derivatisation with N-phenylmaleimide and tris(2-carboxyethyl)phosphine (TCEP) reduction for total thiols.
The analytical system consisted of a 1,290 Infinity II UHPLC hyphenated to a 6470B Triple Quadrupole (Agilent Technologies, Santa Clara, CA, USA). Analytes were separated on a Hypersil gold column (1.9 μm, 100 mm x 2.1 mm) (ThermoFisher, Waltham, MA, USA) with a 40°C oven temperature and total run time of 10 min. The mobile phases were composed of (A) water with 0.1% formic acid and (B) methanol with 0.1% formic acid. The gradient started at 20% of B, was increased to 80% over 4 min, to 99% in 2 min, held for 1 min, then decreased to 20% in 1 min and held for 2 min at a flow rate of 0.5 ml/min. Source parameters were as follows: gas temp was 230°C, gas flow was 10 l/min, nebulizer at 3.79 bar, sheath gas temp at 300°C, sheath gas flow at 12 l/min, capillary voltage at 3000 V in positive mode, nozzle voltage at 1000 V and positive Delta EMV set to 200 V. Ionization was carried out using positive electrospray (ESI+) and detection was made using Multiple Reaction Monitoring (MRM). Quantification and qualification transitions are described in Table S2.

Testosterone and corticosterone quantification was determined using Agilent’s Infinity II UHPLC in line with a 6495 triple quadrupole mass spectrometer and MassHunter workstation software (8.0.8.23.5). Briefly, DSRCT xenograft and PDX samples were homogenized using water containing internal standard (Cerilliant, T070) extracted with tert-butyl methyl ether (Sigma 34875), dried under nitrogen, and derivatized using hydroxylamine hydrochloride (Sigma 431362). The recovered ketoxime steroids were reconstituted in methanol/water (1:1 v/v) and injected into the Infinity II UHPLC. Ketoxime steroids were separated using a Chromolith reverse-phase column (RP-18 endcapped 100–2 mm, Sigma 152006) and introduced into a JetStream source (Agilent) for triple quadrupole analysis. Data were analyzed and quantified using MassHunter software (Agilent)^{39 (link),40 (link)}.