Accq fluor borate buffer

Heparin plasma samples were analyzed for BA using a previously published HPLC method (6 (link)). In short, heparin plasma samples were deproteinized using a 1:9 ratio of 35% sulfosalicylic acid. Plasma supernatant was mixed with ACCQ Fluor Borate buffer and Fluor reagent from the AccQtag Chemistry kit (Waters sa-nv, Belgium) in a 1:7:2 ratio. Standard solutions of BA were treated similarly before HPLC analyses. The derivatized samples were applied to a Waters Alliance HPLC system with the following parameters: XBridge BEH column (4.6 × 150 mm, 2.5 μm; Waters) heated to 37°C; fluorescence detector (excitation/emission wavelength: 250/395 nm); using a flow gradient containing different amounts of buffer A (10% eluent A [Waters], 90% ddH2O), buffer B (100% acetonitrile), and buffer C (100% ddH2O) at a flow rate of 1 mL.min⁻¹.

For the variation of amino acid concentrations in DM media, samples were subjected to an AccQ-Fluor Reagent (AQC, 6-aminoquinolyl-N-hydroxysuccinimide carbamate) derivatization (Waters Corp., Milford, MA, United States) according to the manufacturer’s protocol. AQC was reconstituted at a final concentration of 10 mM in acetonitrile, included in the AccQ-Fluor Reagent Kit (Waters Corp.). Briefly, 10 μL of samples were derivatizated with 70 μL of AccQ-Fluor Borate Buffer (Waters Corp.) and 20 μL of reconstituted reagent. The samples were heated to 55°C for 10 min. The amino acids content was analyzed using an HPLC (PU-1580 Intelligent HPLC pump, Intelligent Fluorescence Detector FP-1520 and Intelligent Sampler AS-2055 Plus, with 10 μl loop; Jasco Corp.). Separation of amino acids was obtained using AccQ-Tag^TM column (3.9 mm × 150 mm) for amino acid analysis (Waters Corp.). A gradient elution was performed maintaining a column temperature of 30°C and using two mobile phases: A (100 ml of AccQ-Tag Eluent A concentrate (Waters Corp.), diluted 1:10 with H₂O for chromatography (Sigma-Aldrich, St. Louis, MO, United States) and B (60% acetonitrile and 40% H₂O for chromatography) (Sigma-Aldrich, St. Louis, MO, United States) with a flow rate of 1 ml/min. The fluorescence detector was set at excitation wavelength of 250 nm and emission wavelength of 395 nm.

Quantitative analysis of hydroxyproline in mouse LVs, a measure of collagen protein content, was performed using high-performance liquid chromatography (HPLC) with the AccQ-Fluor kit (Cat # WAT052880, Waters, MA, USA) according to manufacturer’s protocol. 15 mg tissue was homogenized in liquid nitrogen, hydrolysed overnight in 6 M HCl at 112 °C, dried and derivatized with the AccQ-Fluor borate buffer (Waters). The derivatives were separated using reversed-phase HPLC with the Ultimate 3000 system (Nerliens Meszansky, Oslo, Norway), and quantified by fluorescence detection, with hydroxyproline standards (Fluka, Buchs SG, Switzerland).