A research group wants us to identify and quantify ergosterol in Yeast Cell Pellets. We have recently done regular cell pellets and neuron cell pellets which contained cholesterol. We are having a hard time switching and identifying ergosterol which in turn we cannot quantify it. We do convert the sterols into TMS derivatives and run them on a Rxi-5ms column in a GC-MS. When we obtained an Ergosterol standard we made up a solution at 1.0 mg/mL and ran this thru our prepping process to obtain the TMS derivative. We were able to obtain the peak and it was identified as TMS-Ergosterol from which we were able to obtain the ions we needed to run in SIM mode. We then proceeded to make up a calibration curve for Ergosterol ranging from 0.5 mg/mL down to 0.031 mg/mL. When we ran these on the instrument after running them thru our prep process we were obtaining what appeared to be double peaks or combined peaks for the ions we choose to monitor. When we ran these on the GC-MS in TIC only the 0.5 mg/mL was able to obtain the correct identifier of TMS-Ergosterol and the lower levels were identifying the peak as Neoergosterol acetate and Isoneoergosterol acetate. We decided to run non-derivatized standard thru the GCMS to see if it could identify them at Ergosterol, which it did not. It kepts saying that we ahd 3,5-cyclo-6,8(14),22 ergostatriene. We do have to derivatize the samples due to we are looking at some other sterols (which are coming back good still in both samples and standards) that only work when we derivatize.

Why would the same method we used to do cholesterol and the other sterols in the Cell Pellets not work for ergosterol in Yeast Pellets or even in the standards? Any help would be appreciated.

A quick simplified write up of our prep method is: