2.3.2. HPLC Methods

The HPLC system consisted of a Shimadzu (Kyoto, Japan) LC-20AT pump paired with an SPD-M20A photodiode array detector. The flow rate was 1 mL/min and a 20 μL injection volume was used for each of the methods employed. In the method used initially for indigoids, separations were conducted at controlled temperature (25 ◦C using a heating tape plugged into a variable AC source, monitored with a thermocouple) on an ACE Equivalence 5 C18 150 × 4.6 mm analytical column (VWR, Radnor, PA, USA) attached to a C18 guard column. The analytical column used for separation of Solution III preparations was a Discovery C18 column from Supelco (Bellefonte, PA, USA, 25 cm × 4.6 mm and 5 μm particle size) without temperature control. For the indigoid method, deionized water (solvent A) and acetonitrile (ACN, HPLC grade from Sigma-Aldrich, St. Louis, MO, USA, as solvent B), each at 0.1% (*v/v*) TFA served as mobile phases. The program for separation of the indigoids was 15% B for 5 min, followed by a linear gradient to 50% B in 25 min, then to 70% B in 10 min, followed by 90% B in 10 min held for 10 min (total run time 60 min); re-equilibration time was 15 min. For the Solution III preparations in methanol/HCl (after Manhita et al. [32]), the mobile phase consisted of acetonitrile (A) and 2.5% (*v/v*) aqueous acetonitrile with 0.5% (*v/v*) formic acid (B) in a gradient of 0–100% A from 0–10 min, 100% A from 10–15 min and a 5-minute rinse of 100% solvent B between runs. UV-vis spectra were acquired in the range of 200–650 nm with a resolution of 4 nm. Data were processed using Shimadzu LabSolutions software.
