Ph.Eur. Prednisolone - Related Substances

Discussions about methods, troubleshooting and best practices across both pharmaceutical and biopharmaceutical analysis.

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Hi all,

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.
Some useful info can be found here:
https://extranet.edqm.eu/4DLink1/4DCGI/ ... w/mono/353
Column: Venusil AQ C18.
vmu wrote:
Some useful info can be found here:
https://extranet.edqm.eu/4DLink1/4DCGI/ ... w/mono/353
Column: Venusil AQ C18.


Thanks, I have been looking at the knowledge base and came across the column they referenced. However would a particular brand make such a difference?

It's something we haven't tried yet though. Another option we're considering is if a H-Class UPLC might help due to a smaller dwell volume.
I don't think the dwell volume is the cause since the problem peak pair is eluted in the first isocratic part
of the run. However, insufficient equilibration time before the next run may cause problems with the separation. Check this.

Also check your data collection rate. The rate might be too low.
Philip01 wrote:
However would a particular brand make such a difference?

The column in the monograph info is an "AQ" column. This can change selectivity enough in comparison with Luna C18.

You can also try to decrease the injection volume if the detection sensitivity is sufficient.
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