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- Posts: 5
- Joined: Sat Jan 18, 2020 11:54 am
We're following the Ph.Eur. monograph for Prednisolone but are having trouble in meeting the peak-to-valley ratio between Prednisolone and Impurity A (Hydrocortisone) for the Related Substances test.
The column detailed within the monograph is:
Length: 15cm
Internal Diameter: 4.6mm
Stationary Phase: C18 (End-Capped) 3μm
Instrument Parameters:
Mobile Phase A: Water
Mobile Phase B: ACN, Methanol (50:50 v/v)
Column Temp: 40 degrees
Flow Rate: 1ml/min
Injection volume: 10μl
Gradient table:
T0 - T14 (MPA 60% and MPB 40%)
T14 - T20 (MPA 60 -> 20% and MPB 40 -> 80%)
T20 - T25 (MPA 20% and MPB 80%)
The reference solution is a CRS vial containing these impurities made up in solvent mixture, Acetonitrile, Water (40:60). The requirements of the monograph is to have a peak to valley ratio of not less than 3.0 which we've been unable to meet. We're running on a Waters Alliance HPLC with a Luna C18 column (new) and are only able to get a p/v ratio of ~1.8 at best. We've also tried changing some of the instrument parameters that are allowed such as increasing the column temperature which did help a little.
Any advice would be greatly appreciated, thanks.