2695 separations module

Cells were fixed with PFA as described above for adipocytic quantification. After washing, 500µl of PBS was left in the wells, plates were sealed with parafilm and stored at 4°C until processing for HPLC analysis. Cells were detached from the plate with 1mL (2 x 0.5ml) of PBS and lipids extracted with chloroform/methanol (2:1; v/v) under agitation for 30min at room temperature. The resulting lipid extract was then subjected to saponification, followed by fatty acid derivatization into naphthacyl esters as described previously (46 (link)). Fatty acid derivatives were applied into the HPLC Alliance system (2695 Separations Module, Waters, Saint-Quentin-en-Yvelines, France) equipped of an autosampler (200ml-loop sample), a photodiode array detector (model 2998), and the Empower software for data analyses. Fatty acid derivatives were eluted on a reverse phase YMC PRO C18 column (3mm, 4.6x150mm, 120Å) by using two solvent systems: a) methanol/acetonitrile/water (64:24:12; v/v/v) and, b) methanol/dichloromethane/water (65:28:7; v/v/v) under identical conditions reported previously (46 (link)). Fatty acid derivatives were detected at 246 nm and quantified by an external standard curve realized with the naphthacyl 19:0 derivative. Note that 19:0 was also used as internal standard to evaluate FA recoveries. Lipidomics LC-HRMS standards and solvents are provided in Table S1.

The concentrations of ZEN and its derivatives were determined by HPLC with the use of the Waters 2695 Separations Module, Waters 2475 Multi λ Fluorescence Detector and Waters 2996 Array Detector. Excitation and emission wavelengths were 274 and 440 nm, respectively. The reverse-phase column was C-18 Nova Pak (3.9 × 150 mm), and the mobile phase was acetonitrile-water-methanol (46:46:8, v/v/v) at a flow rate of 0.5 mL·min⁻¹. The mycotoxins were quantified by measuring the retention time of the peaks based on the relevant calibration curves (correlation coefficient of 0.9996 for ZEN, 0.9989 for α-ZEL and 0.9976 for β-ZEL). Detection limits were determined at 0.001 µg·g⁻¹ for ZEN, 0.003 µg·g⁻¹ for α-ZEL and 0.002 µg·g⁻¹ for β-ZEL.

The hydrolysis of bradykinin to des-Arg-bradykinin (Pro-Pro-Gly-Ser-Pro-Phe-Arg) catalyzed by wild-type or mutant hcAMPP, was carried at out for 15 min at 37°C using 50 mM Tris-HCl pH 7.5 as buffer, and 0.5 mM MnCl₂ as cofactor. Reactions were terminated by heating the reaction mixture at 100°C for 10 min prior to cooling on ice. After centrifugation for 2 min, 25 μL aliquot of the supernatants were analyzed by using a reversed-phase HPLC system (Waters 2695 Separations Module) equipped with an autosampler, a UV-Vis photodiode array detector (215 nm monitoring), and a Vydac C18 column (5μm, 25 cm × 4.6 mm). Column elution was carried out over the course of 1 h with a gradient running from 30 to 70% (v/v) methanol in H₂O containing 0.1% (v/v) trifluoroacetic acid. Initial velocity data were measured at varying bradykinin concentration (20–1200 μM) and then fitted using the Michaelis-Menten equation to define the steady-state kinetic constants k_cat and K_m:
$v=\frac{Vmax\left[S\right]}{Km+\left[S\right]}$
where ʋ is the initial velocity, K_m is the Michaelis constant, V_m is the maximum velocity and S is the substrate (bradykinin) concentration. For determination of the k_cat and K_m for manganese activation, the concentration of MnCl₂ was varied from 1–400 μM at fixed bradykinin concentration.

The chromatographic analysis of lactoferrin was carried out on a HPLC system (2695 Separations Module, Waters; Milford, MA, USA) coupled with a photodiode array detector (PDA 2996 detector, Waters; Milford, MA, USA). Separation was performed using a Symmetry C4 Column (300 Å, 5 μm, 4.6 mm × 250 mm, Waters). Acetonitrile (eluent A) and 0.1% trifluoroacetic acid in water (eluent B) were used as mobile phase. The flow rate was set at 1.0 mL/min and the LC elution gradient was as follows: initial 30% A, 5 min 55% A, 10 min 60% A, 12 min 30% A and hold on for a further 4 min for re-equilibration, giving a total run time 16 min. The column temperature was kept at 25 °C and the injection volume was 50 μL for standards and sample solutions. The wavelengths was set at 280 nm for detection. Waters Empower 2.0 chromatography software package was used for HPLC system control, data acquisition and management.

The HPLC determination of DMAT was performed using the Waters 2695 Separations module equipped with a thermostable autosampler (5 °C), a column module (35 °C), and a Waters 2996 diode array detector (DAD). The separation of the compounds was achieved using a VDSphere PUR C18-M-SE (4.6 × 150 mm, 5 µm) (Agilent technologies, Palo Alto, CA, USA) column. For HPLC-MS measurements, the HPLC instrument was coupled with a MicroTOF-Q-type Qq-TOF MS instrument equipped with an ESI source from Bruker (Bruker Daltoniks, Bremen, Germany). The flow rate and run time were 1.0 mL/min and 12 min, respectively. The active component DMAT was detected with DAD at 260 nm and MS. For the separation of the compounds, isocratic eluent composed of 25% methanol, 40% AcN/water (9/1) containing 0.1% trifluoroacetic acid (TFA), and 35% water was used.