Gradient elution was performed as
follows: 0–5 min, 5–9% phase B; 5–15 min, 9% phase B; 15–22 min,
9–11% phase B; 22–38 min, 11–18% phase B; 38–43 min, 18–23%
phase B; 43–44 min, 23–90% phase B; 44–45 min, 90–80% phase
B; 45–55 min, isocratic at 80% phase B; 55–60 min, 80–5% phase
B; and a re-equilibration period of 5 min with 5% phase B used
between individual runs.