996 photodiode array

The NTA-NTBI method previously described¹⁹ (link) was adopted with minor modifications. Briefly, 0.02 mL of 800-mM NTA (at pH = 7) was added to 0.18-mL serum and allowed to stand for 30 minutes at 22^°C. The solution was ultrafiltered using Whatman Vectaspin ultracentrifugation devices (30 kDa) at 12320g and the ultrafiltrate (0.02 mL) injected directly onto an high-performance liquid chromatography column (ChromSpher-ODS, 5 μM, 100 × 3mm, glass column fitted with an appropriate guard column) equilibrated with 5% acetonitrile and 3-mM deferiprone (DFP) in 5-mM MOPS (pH = 7.8). The NTA-iron complex then exchanges to form the DFP-iron complex detected at 460 nm by a Waters 996 photodiode array. Injecting standard concentrations of iron prepared in 80-mM NTA generated a standard curve. The 800-mM NTA solution used to treat the samples and prepare the standards is treated with 2-μM iron to normalize the background iron that contaminates reagents. This means that the zero standard gives a positive signal because it contains the added background iron as an NTA-complex. When unsaturated transferrin is present in sera, this additional background iron can be donated to vacant transferrin sites resulting in a loss of the background signal and yielding a negative NTBI value.

HMF and furfural were analyzed with Waters 2695 separation module and Waters 996 photodiode array (PDA) detector. An Atlantis T3 (3 μm, 4.6×150 mm) or Atlantis dC18 column (5×μm, 4.6×150 mm) was used when HMF and furfural were determined either from the organic phase or from DES, respectively. A mixture of water (0.1 % TFA) and methanol (0.1 % TFA) (90 : 10) was used as the mobile phase, with a flow rate of 1 mL min⁻¹. The column temperature was kept constant at 30 °C. UV detection for HMF was performed at 284 nm, while for furfural, 277 nm was used as wavelength. Calibrations were performed using commercial HMF and furfural, and all samples were analyzed as duplicates. Yields are expressed as the ratio of furfural or HMF obtained in the organic phase to the initial xylose or glucose fed to the reaction.

A volume of 10 mL of the culture supernatants was purified through C18 Maxi-Clean 10 g cartridges (Alltech). Compared to the lipopeptide HPLC analysis protocol, all the volumes used were adapted. A sample of 100 μL of the lipopeptide mixture concentrated at 250 mg/L was charged in the HPLC (Online Degaser, 717 Autosampler, 660S Controller, 626 Pump, 996 PhotoDiodeArray, Waters Corp., Milford, MA, USA). The column used was a C18 (5 μm, 300 × 10 mm, ACE), working at a flow rate of 3 mL/min. The mycosubtilins were separated with an acetonitrile–water–trifluoroacetic acid solvent, 35:65:0.1, v/v/v for 54 min, followed by a 6 min rinsing step with acetonitrile–trifluoroacetic acid solvent, 100:0.1, v/v. As for lipopeptides analysis, the retention time and second derivative of the absorption spectrum between 200 and 400 nm were used to identify the eluted molecules (Millenium 32 Software, Waters).