The solvents and optimized gradient conditions for LC were: Solvent A, 50% methanol/50% water/0.05% formic acid; Solvent B, 90% acetonitrile with 5 mM ammonium acetate; elution gradient: 0 min–100% B; 20–25 min –40% B; post-run time for equilibration, 10 min in 100% B.