I am trying to extract the sphingolipid d18:1 sphingosine from mammalian red blood cells and quantify it by HPLC-Fluorescence. I have tried to use the lipid extraction method in this paper (Article Quantitation of free sphingosine in liver by high-performanc...
) By comparing the fluorescence signal of internal standard d17:1 sphingosine in an extracted sample to a non-extracted neat sample, my extraction efficiency is about 10%. The one liberty that I did take with the method is that I have used 10 microliters of red blood cells which is about 100 million cells. Does anyone have any ideas for why I am getting such low extraction efficiency? Is it possible that I am using too many cells and am overloading the extraction process? Thank you.
Michal Surma Thank you for your response.
By "non-extracted neat standard", I mean a pure sample of the lipid that I analyze without extraction. To be more concrete, I buy d17:1 sphingosine in powder form and make a stock solution (e.g. 1 mM in methanol). Then, I measure the fluorescence signal of, say, 100 pmoles of d17:1 sphingosine from that stock solution that hasn't been through the extraction procedure and 100 pmoles of d17:0 sphingosine after it has been extracted. The fluorescence signal of the extracted d17:1 sphingosine is typically about 10% that of the signal of the non-extracted d17:1 sphingosine.
Based on your comments, I think one thing I should try is shaking the extraction for a longer period of time. The paper I referenced did not explicitly state a shaking time, so I only vortex for a few seconds and then separate the two phases. I will try shaking for 30 minutes before separating the phases. Thank you again.
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