Hplc instrument

The HPLC instrument (Dionex) was equipped with a HP 1050 multidiode array detector, a 20 μL loop and a 250 × 4.6 mm, 5 μm Thermo Scientific Hypersil GOLD column. Two solvents were used for elution; A (water-TFA 99.5:1; v/v) and B (acetonitrile-TFA 99.5:1; v/v). The elution profile of the applied HPLC gradient is shown in Fig. S1. The analytical HPLC pump system was purged with both solution A and solution B for 15 min each with a flow of 5 mL/min. The column was thereafter equilibrated with a flow of 1 mL/min in 30 min with acetonitrile-super distilled water (10:90 v/v). 20 μL of each sample was injected with an autoinjector. The elution profiles consisted of isocratic elution (10% B in A) for the first 4 min, followed by linear gradient (10% B-14% B) for the next 10 min, isocratic elution (14% B) for the next 4 min, linear gradient (14% B-18% B) for the next 10 min, linear gradient (18% B-28% B) for the next 10 min, linear gradient (28% B-40% B) for the next 4 min and isocratic elution (40% B) for 10 min. The flow rate was 1 mL/min.

The HPLC system consisted of a Waters Alliance e2695 separation module and 2998 photodiode array detector (PDA). Data acquisition was performed using the Empower software (version 2.0).
A Dionex HPLC instrument was also used for intermediate precision measurements. The HPLC apparatus consisted of a Dionex P580 LPG pump, an ASI-100 T autosampler, an STH 585 column oven, and a PDA-100 UV detector; data were acquired and processed by a Chromeleon Data System (Dionex Corporation, Sunnyvale, California, US).