Separation Variable Value
Column Zorbax Eclipse Plus C18 column (250 × 4.6 mm, 5 μm)
Mobile phase 0.1% formic acid (A) and Acetonitrile (B)
Diluent Acetonitrile and water (50:50)
Separation mode Gradient mode
  Time 0.1% formic acid (A) ACN (B)
0 min 60% 40%
1 min 60% 40%
10 min 10% 90%
18 min 60% 40%
20 min 60% 40%
Flow rate 1.0 mL min-1
Temperature Ambient
Samples size 20 μL
Detection wavelength 252 nm
Retention time 4.82 min
Table 1: Separation variable of RP-HPLC method for AMD.