Ph.Eur. Hycrocortisone related substances analysis

[Anny]

Does anyone have experience on which column to use in EP Hydrocortisone related substances analysis?

The monograph calls for a 250x4,6 mm column with base-deactivated end-capped C 18 silica 5um. We have used Hypersil BDS C18. The isocratic mobile phase is 22% tetrahydrofuran in water at 1 ml/min. T = 45 C. Detector set at 254 nm. The problem is poor resolution between predisolone and hydrocortisone (sample in mobile phase). The monograph requires 2.2 the best we can get is 1.77. We have made variations in the column, temperature and mobile phase.

If you have any suggestions to improve the resolution under these conditions, I would be very grateful to hear of them!
Anny

[Bryan Evans]

That separation was done on our Cadenza CD C18 column
(with a resolution of 2.7):

http://www.imtakt.com/TecInfo/TI039E.pdf

If you email me, we will be more than happy to help you
out - thank you.

[Mark Tracy]

I used the conditions in McWhinney, et.al., Clin.Chem. 1996, 42, 979-981. (MeOH:THF:H2O 3:25:72) with good results. Prednisolone/Cortisol resolution 2.6. You can see it at http://www1.dionex.com/en-us/acclaim_li ... 29719.html
I have also seen that several commercial clinical diagnostic labs use the same method.

[Albany-12303]

Ask pharmeuropa (EP)



http://www.pheur.org/site/page_630.php?rubrique=228

[bartjoosen]

Or search in the EP database:
http://extranet.pheur.org/publications/ ... s_sw.shtml

[Mark Tracy]

I tried the two EP links. A search for "hydrocortisone" gave a link to monograph 335. The online help page gave no hints, no column suggestions, no reference chromatogram, no additional pdf, only the catalog numbers for reference standards. They really want you to purchase their books!

[bartjoosen]

Try prednisolone, same column, same mobile phase, same resolution requirements.

[Anny]

Thanks everybody for your valuable comments!

Special thanks goes to bartjoosen: We tried the Kromasil C18 suggested by Ph.Eur. in the prednisolone monograph: resolution 2.8! Problem solved!

Guess we learned yet another lesson in using the pharmacopoeia!

[Bruce Hamilton]

Hi,

The EP have responded to my email pointing out that their knowledgebase said a Lichrosorb Si column was used for the hydrocortisone related substances test. Rather difficult to use, when the monograph specified a 250mm x 4.6mm 5um base-deactivated end-capped octadecylsilyl silica gel.

They say the entry was a mistake, will be corrected, and that an Alltima BDS column was used for the last requalification. I know the original poster has solved their problem, but in case anyone else wants to try...

Bruce Hamilton