I lose all my surrogate standards in my samples and blank samples (prepared with mixtures and same amount of surrogates). A while ago, I was suspicious of volume reduction methods I use (rotary evaporator and N2 blowdown) and I was right to be. After fixing it, I continued to move backwards and fixed couple of problems with column clean-up too. Yet I still can't find the proper way to achive seeing concernations of spiked standards.

The lipid matrix I use is blood serum. I inject the standards to the samples and leave overnight and +4 °C. Next day I start with MTBE+Hexane solution and formic acid and centrifuge for 10 mins at 2500 rpm. When it's done, you can clearly see the upper hexane phase. After separating the upper phase and collecting it somewhere else, I add more hexane (as much as volume of the blood serum I used) and do this step twice more. In the end, I have upper hexane phase colected three times.

Then after adding H2SO4 to the collected upper hexane phase to get rid of the possible lipids caught in, I separete the upper phase again and do a volume reduction. After that comes the column clean-up.

I really wonder what other possible ways to do extraction or what can you suggest me to do. I think I can start with increasing centrifuge time.