extraction of steroids in oil suspension

[dmaa]

Im struggling with being able to extract testosterone cypionate and similar steroids from oil based suspensions. I have tried extracting with ethyl acetate, blowing down under nitrogen, and resuspending in ACN, all I saw was benzyl alcohol.

I tried the below method from the paper "HPLC method development for testosterone propionate and cipionate in oil-based injectables"

"Ten ampoules of each product containing TP or TC were
mixed, and 200l of them were added with 0.1 M SDS
to 10 ml (TP) or 25 ml (TC). These mixtures contained
500g ml−1 (TP) or 1000g ml−1 (TC). They were vigorously shaken for 5 min, sonicated for 15 min and centrifuged
for 5 min to separate the oil from the aqueous phase. The
aqueous phase was removed (without disturbing the oil) and
used to prepare 5 g ml−1 TP or TC solutions using 0.1 M
SDS (100% TP or TC). The recoveries found for TP and TC
were close to 100%, respectively (see Section 4.1). After extraction and for quantitation purposes, samples were added
with MT(IS) for TP or BLS(IS) for TC to obtain 5 g ml−1 of
IS. Finally, these solutions were injected in the HPLC system
(20l)."


When I tried this method I had horrible recovery. If I interpreted it right, I added 200ul of the oil based suspension which was 200mg/ml to 10ml of 0.1M SDS. I vortexed, sonicated, then centrifuged the sample. I then carefully pipetted the SDS solution into the autosampler vial. Great recovery on the preservatives but barely any analyte of interest.

Im now trying the sds page method above but adding ACN at the last step, along with salts to create 2 phases. I will run both the acn and sds phase.

Any other suggestion on a viable extraction method? My mobile phase h20 0.1% TFA, and ACN 0.1% TFA. Using a c18 column detection at 245 nm

[Hollow]

don't know how well it will work, but you may try to dissolve an aliquote of your oil-based sample in heptane, then load that onto a bare silica (normale phase) SPE cartride, wash with heptane to remove all/most of the oil, then elute with heptane+ethyl acetate (e.g. 50:50), or pure EtAc or isopropanol or similar.

You may even try that approach first on TLC plates to get an idea of how much heptane can be used to wash the oil and how your steroids behave (e.g. what Rf value will they have with pure heptane --> retention factors --> column volumes needed for elution).

[LuccasLN]

Hi dmaa,

I will agree with Hollow and say that the best way would be SPE. but just in case, have you tried using methanol or a water-methanol mixture to do this extraction in similar volumes? maybe methanol is polar enough to separate phases! of course you would have to evaporate methanol to avoid any reaction with TFA.