I work at Antidoping Laboratory at Jockey Club of Sao Paulo - SP - Brazil.
We are working with the prednisone and prednisolone detection by HPLC-UV, but we are having lots of difficulties in the separation of the prednisone and prednisolone peaks. I am using a LiChrospher 100 RP-18 125 x 4 mm (5 µm) column. I tryed lots of mobile phases:
CH2Cl2: acetic acid 0,05% : methanol
CH2Cl2: acetic acid 0,05%
acetonitrile : 0,02% triethylamine
methanol : 0,02% triethylamine
acetic acid 0,05% : methanol
None of them gave me a good separation between these two compounds. Dou you have any other suggestion or reference that could help me on this ???

Best regards

Maristela H. Andraus
dcpa_jcsp@uol.com.br

Maristela,

The current BP has a related substances method for prednisone. It quotes a 250 x 4.6mm ODS column at 45C, flow rate 2.5ml/min (a bit high, but it must work!). The mobile phase is 10% acetonitrile/20% methanol/70% water. The prednisone and prednisolone peaks elute at approx. 19 and 23 minutes. As this is a related substances method, there is a lengthy gradient run starting at 25 minutes, with mobile phase B as pure ACN, but as your two analytes have eluted by this time this might at least give you a few ideas.

I am surprised that in a anti-doping lab that you wouldn't be using LC/MS or better still LC/MS/MS. I would think that positive compound identification would be vital.

A.A.