Hi,

I’m in need of some advice on the best column to use to resolve some steroid hormones of interest in our lab. Specifically, I’m looking to develop a GC/MS method of quantitation for the following steroid hormones from mouse and canine plasma:

5-AED (androstenediol; 5-androsten-3b,17b-diol);
DHEA (dehydroepiandrosterone; 5-androsten-3b-ol-17-one);
Corticosterone ( 4-pregnen-11b,21-diol-3,20-dione);
Cortisol (hydrocortisone; 4-PREGNEN-11â, 17, 21-TRIOL-3, 20-DIONE)
Estradiol (1,3,5(10)-estratrien-3,17a-diol);
Testosterone (4-androsten-17b-ol-3-one);
and eventually DHEA-S (5-androsten-3b-ol-17-one sulfate)

Currently, I’m using a Restek Rtx-5MS column (30m x 0.25mm x 0.25mm) and I’m not getting the resolution I’d like to see. When I run pure standards of DHEA, 5-AED, estradiol, testosterone and corticosterone what I see is DHEA and 5-AED not baseline resolved and a co-eluting peak of estradiol and testosterone. I’ve tinkered with the temperature gradient by changing either the ramp rate or inserting temperature holds to try and resolve these compounds but to no avail. All I do is either shorten or lengthen the run but w/o improvements in resolution. Thus I’ve come to the conclusion that it’s the column that may be at issue. However, I’m not at expert in GC/MS so I’m turning to pro’s for help. If you’ve got experience with these compounds and have a column or other suggestion, I’d be most appreciative in hearing from you.

Sincerely,

Jimmy Pendergrass

Are you assaying as-is or as trimethylsilyl derivatives?

James,
You might find useful information in a paper that I recently read, a review titled "Separation and detection of neuroactive steroids from biological matrices".
(Journal of Chromatography A, 955(2)151-182)