The applied separation voltage was 30 kV with positive polarity on the injection end. The comparative method, using the LC/MS/MS analysis, Bax protein was

performed on chromatographic equipment consisting of a high-performance liquid chromatography (HPLC) system (Agilent Technologies – Germany). Separation was performed on an Atlantis HILIC Silica Column (30 mm, 2.1 mm ID, 2.0 μm particle size) Waters. A multi-step isocratic and linear gradient of solvent A (H2O + 0.1% formic acid) and B (95:5 acetonitrile/H2O + 0.1% formic acid) was applied. The runs were performed using a mobile phase as follows: 0–2.5 min, 90% solvent B (isocratic mode); The flow rate was set at 0.15 mL/min. In all instances, the injection volume was 0.5 μL.

The column temperature was set to 30 °C. The LC system was coupled to a mass spectrometer system consisting of a hybrid triplequadrupole/linear ion trap mass spectrometer Q Trap 3200 (Applied Biosystems/MDS Sciex, Concord, Canada). Analyst version 1.5.1 was used for the LC/MS/MS system control and data analysis. The mass spectrometry was tuned in the negative and positive modes by infusion of polypropylene glycol OTX015 mw solution. The experiments were performed using the TurboIonSprayTM source (electrospray-ESI) in positive ion mode. The capillary needle was maintained at 5500 V. MS/MS parameters: curtain gas, 10 psi; temperature, 400 °C; gas 1, 45 psi; gas 2, 45 psi; CAD gas, medium. Others parameters for the cone and collision energy are listed Bacterial neuraminidase in Table 1. HMF was monitored and quantified using multiple reaction monitoring (MRM). Optimisation of the mass spectrometer was performed by the direct infusion of an aqueous solution containing HMF investigated here. All reagents were of analytical grade, solvents were of chromatographic purity and the water was

purified by deionisation (Milli-Q system, Millipore, Bedford, MA, USA). 5-HMF, caffeine, sodium tetraborate (STB), methanol (MeOH) and sodium dodecylsulfate (SDS) were obtained from Sigma–Aldrich (Santa Ana, CA, USA). Sodium hydroxide was purchased from Merck (Rio de Janeiro, RJ, Brasil). Stock solutions of 5-HMF (1000 mg L−1) were prepared in MeOH:water (50:50, v/v) at a 1000 mg L−1 concentration and stored at 4 °C until analysis. Separate aliquots (0.1, 0.2, 0.4, 0.6 and 0.8 mL) of 5-HMF stock solution were transferred to a 10 mL volumetric flask and diluted with distilled water to make the concentrations: 10, 20, 40, 60 and 80 mg L−1, respectively. Caffeine was used as internal standard (IS), and stock solutions (1000 mg L−1) were prepared by dissolving 100 mg of caffeine in 100.0 mL of deionised water and stored it at 4 °C until analysis. The standard working solutions were prepared every day. In the direct analysis of 5-HMF the optimal electrolyte was composed of 5 mmol L−1 STB and 120 mmol L−1 SDS at pH 9.