Spherisorb ods2 c18

The content of thiols was determined using aliquots of 25 μl supernatant previously extracted with 0.1 M HCl and neutralized by adding an equal volume of 0.1 M NaOH. The extraction and quantification of thiols followed the protocol described by Schupp and Rennenberg (1988) (link) and modified by Strohm et al. (1995) (link). Thiols were derivatized with monobromobimane (5 μl, 100 mM) and stabilized by adding 100 μl acetic acid (9%, v/v) after 15 min of derivatization in the dark at 37°C. Aliquots of 100 μl derivatized solution were injected into an HPLC system (Dionex UltiMate 3000; Thermo Fisher Scientific GmbH; Waltham, MA, United States) and thiol derivatives were separated on a Spherisorb ODS2 (C₁₈; 250 × 4.6 mm, 5 μm particle size) column (Waters Corporation, Milford, CT, United States) using a solution of 0.25% (v/v) acetic acid and 10% (v/v) methanol as buffer A and 0.25% acetic acid and 90% (v/v) methanol as buffer B. Thiols were detected fluorometrically (Waters 474 Fluorescence detector, Waters Corporation, Milford, CT, United States) with excitation at 390 nm and emission at 480 nm. The [³⁵S]SO₄^2- incorporation into thiols was determined by a radioactivity detector module (Radio Flow Detector FlowStar LB 513; Berthold Technologies GmbH & Co. KG, Bad Wildbad, Germany) connected to the HPLC.

Chromatographic analyses were
performed on a 1260 Infinity HPLC system (Agilent Technologies, Waldbronn,
Germany). The system consisted of an online vacuum degasser, a quaternary
pump, a thermostated column compartment and a ultraviolet–visible
(UV–vis) detector with variable wavelengths. OpenLAB CDS Rev.
C.01.05 v.37 software was used for system control and data acquisition.
Different analytical columns used for HPLC studies were tested. RPLC
columns: Symmetry C₁₈ (150 × 3.9
mm²; particle size 5 μm), Spherisorb ODS2 C₁₈ (250 × 4.6 mm²; particle size 5 μm) from Waters
(Milford MA), Luna C₁₈ (150 × 3.9 mm²;
particle size 5 μm) provided by Phenomenex (Torrance), LiChrospher
RP-18 (150 × 4.6 mm²; particle size 5 μm), and
a HILIC column, SeQuant ZIC-HILIC (150 × 3.9 mm²;
particle size 5 μm) were purchased from Merck KGaA (Darmstadt,
Germany).
After several optimization studies, LiChrospher RP-18
was chosen as the preferred option due to its better chromatographic
performance with the iron chelate investigated. The mobile phase selected
was composed of a mixture of acetonitrile and sodium borate buffer
0.20 mM (pH = 8) (70:30, v/v) applied at a flow rate of 1.0 mL/min
in isocratic mode. The injection volume was set at 10 μL. Finally,
measurements were performed at a wavelength of 250 nm after previously
examining the corresponding UV–vis spectra in a spectrophotometer
(Figure S1A).