2 3 4 5 6 pentafluorobenzyl bromide

Methyl tert-butyl ether (MTBE), methanol, chloroform, butanol, heptane, ethyl acetate, sodium chloride, acetic acid, acetone, myristic-d27 acid, Supelco 37-FAME mix potassium hydroxide, hydrochloric acid, hexane, N,N-diisopropylethylamine, 2,3,4,5,6-pentafluorobenzyl bromide and acetonitrile were purchased from Sigma Aldrich. The free fatty acid standard containing 52 fatty acids from C4 to C24 was purchased (Nu-Check Prep, Inc., Waterville, MN, USA). Deionised water (18.2 MΩ) was produced using a Milli-Q™ (Merck KGaA, Darmstadt Germany) system throughout. Glass tubes, 15 mL (Kimble), with Teflon lids and glass Pasteur pipettes were used for extractions; all glassware was rinsed with LC-MS-grade methanol before being heated at 400 °C for 4 h prior to use. A positive displacement pipette with glass tip (BRAND^®, Wertheim, Germany) was used for accurate pipetting. Pre-packed silica-based solid-phase extraction cartridges were used (500 mg, 6 mL SampliQ™, Agilent Technologies, Mulgrave, Australia).

GSH, GSSG, γ-Glu-Cys, Cys-Gly, 5-oxo-proline, Glu and the other amino acids used in the study, CH₃OH, CD₃OD (99.8 atom % ²H), acetone, pentafluoropropionic anhydride, 2,3,4,5,6-pentafluorobenzyl bromide, ophthalmic acid (γ-glutamyl-α-amino-n-butyryl-glycine) and borax (for the preparation of borate buffer) were purchased from Sigma-Aldrich (Steinheim, Germany). Hydrochloric acid (ultrapure, 37%) was from AppliChem (Darmstadt, Germany). Stock solutions were prepared in, and diluted with, deionized water, as appropriate. Glassware for GC-MS (1.8-mL autosampler vials and 0.2-mL microvials) were purchased from Macherey-Nagel (Düren, Germany).
Safety Considerations. PFPA is corrosive and malodorous. PFB-Br is corrosive and a lachrymator. Inhalation and contact with skin and eyes should be avoided. All work should be, and was, performed in a well-ventilated fume hood.

Plasma concentrations of NO metabolites (NO₂⁻ and NO₃⁻), were analysed using previously described methods (Qadir et al., 2013 ). Spiking solution was prepared from 5 mM sodium nitrate-¹⁵N and 0.05 mM sodium nitrite-¹⁵N (Cambridge Isotope Laboratories, Inc. Andover, MA, USA) and used as an internal standard. One-hundred microliters of plasma/sample, 100 μl of spiking solution, 20 μl of 2,3,4,5,6-pentafluorobenzyl bromide (Sigma-Aldrich), and 800 μl of acetone (VWR, Lutterworth, Leicestershire, UK) were pipetted into Falcon™ round-bottom polystyrene tubes and placed in a heating block at 50 °C/120 min. Following incubation, acetone was evaporated under a nitrogen stream for 10 min. Samples were then allowed to cool before 2 ml of toluene (Fisher Scientific UK Ltd) was added to each tube and the tubes vortexed for 15 s. Subsequently, 1 ml of distilled H₂O was pipetted into each tube, and the samples were re-vortexed twice for 15 s with a rest of 15 s in between. Using glass Pasteur pipettes, the top layer was transferred into amber glass vials and stored at -20 °C pending GCMS-analysis. Other variables such as column type and ionisation temperatures were as described by Tsikas (2000) (link).