Luna pfp

Ten milligrams of the methanolic extract of R. schneideri poison was dissolved in 40 µL of amonium bicarbonate completing the volume to 100 µL using trifluoracetic acid (TFA) 0.1% and centrifugated (14000 g, 3 min, 4°C) to remove the insoluble material. The supernatant was used for purification.
The methanolic extract was fractioned by HPLC on a reversed phase Phenomenex Luna PFP (250 x 4.6 mm) with TFA 0.1% + acetonitrile (ACN) 10% as mobile phase and ACN 90% + 0.1% TFA as eluent. Fractions were eluted with a linear gradient 0-65% of eluent. The outflow was monitored at 214 e 280 nm. The fractions were manually collected and then lyophilized.^{9 (link)}

A sample of cadA and cadB (approximately 0.5 mg) were dissolved in 2.0 mL of 6 M hydrochloric acid and hydrolyzed overnight at 110 °C. The hydrolysates were treated with Marfey’s reagent (FDNP-Val-NH₂) as previously reported [31 (link)]. The derivatives were analysed by LC–DAD–MS (column: Luna PFP, 3 μm, 2.0 mm × 100 mm; Phenomenex, Torrance, CA, USA), set at 28 °C. The mobile phase was H₂O containing 0.1% formic acid (A) and ACN (B), at a flow rate of 0.3 mL/min. A linear gradient elution was performed as follows: 25% B (3 min), 25–65% B (3–40 min), 100% B (41–45 min), 25% B (45–50 min).
The same procedure was repeated for cadophorin B (0.2 mg) to confirm the results.

GSH levels were measured in plasma and platelets after 30 min incubation of PRP with and without NAC, exogenous GSH, or GSH ethyl ester (GSHO-Et). Platelets were then isolated by centrifugation (700× g for 15 min) and washed with phosphate buffered saline (PBS). Intracellular and plasma GSH levels were quantified using a LC-MS/MS method previously developed and validated by us [32 (link)]. Briefly, chromatographic separation was conducted on a Luna PFP analytical column (100 × 2.0 mm, 3 µm, Phenomenex, Castel Maggiore, Bologna, Italy), eluted at 35 °C under isocratic conditions at 200 µL/min by 1% methanol in ammonium formate 0.75 mmol/L adjusted to pH 3.5 with formic acid. Analysis was performed by an Accela chromatographic system coupled with a triple quadrupole mass spectrometer TSQ Quantum Access (Thermo Fisher Scientific, Rodano, Milan, Italy) using an electrospray ionization source in positive ion mode. The transitions used in the multiple reaction monitoring were m/z 308.1→m/z 76.2 + 84.2 + 161.9. Data were obtained by comparison with calibration curves using GSH standard solutions (Sigma-Aldrich S.r.l.). The intra- and inter-day CVs % obtained with standard samples were <5%. The limits of detection were 0.031 µmol/L. Levels of GSH were expressed as μmol/L concentration and data are reported as mean ± SD.