Hi!

Have you checked the components of internal supelco FAME standard. If its same as methyl C13:0, or if methyl C13:0 is one of the component of internal standard, then yes, it will certainly superimpose at the same place in the sample and give higher amounts.

Next if methyl C13:0 is present in your sample then why you are using such internal standard that contains it. there's a wide range of methylated fatty acids available with supelco, cayman and avanti polar lipids, you may choose an another internal standard such as C21:0 or C23:0 or anu other.

Good Luck.

Hi Puja,

Thank you for your reply. I have checked and Supelco has methyl C13:0 in it. For my samples I am trying to find an internal standard for it and the protocol states C13:0 so i was confused whether it is the same. I have it cleared up after reading the protocol for the 2nd time. I have found out that the methyl decaoate does not dissolve very well in methanol, hence does not act well as an internal standard. As I will need the readings from C14 -C24 for detecting the amounts of lipids in fish flesh (ref to marine oils). Would you have any idea what internal standards I could use?  I will be getting menhaden oil as a positive control for the relative amounts of each FA.

First run a test sample on a GC-MS identify all the fatty acid methyl esters, purchase a FAME mix representative of your sample as obtained from the GC-MS analysis. Make standard curves from your mix (within the range of your sample). And use this to get absolute measures of the FAME in your sample. Based on the GC-MA analysis choose an odd chain FAME that is not present in your sample eg C13 or 15.. USe the odd chain FAME as an internal standard during your extraction. .

Thanks Thomas. I will note that and add this into my experiment.

Addition of internal standards as reported in previous answer is essential. Have a look to:

Alvaro, D., Angelico, M., Cantafora, A., Di Biase, A., Gaeta, G., Ginanni Corradini, S., et al. (1986). Influence of tauroursodeoxycholic and taurodeoxycholic acids on hepatic metabolism and biliary secretion of phosphatidylcholine in the isolated rat liver. Biochimica et Biophysica Acta (BBA), 878(2), 216-224.

Hi Grace

 As you know, fatty acids with even number of carbons are more common in nature. That is why most people use fatty acids with odd number of carbons as IS. We started using 21:0, but  after having some problems with peaks, we switched to 19:0 and it is perfectly fine. It is important to add your internal standard in a known concentration before you treat your sample. We usually add it as a 5% of the lipid we are subjecting to acid-catalysed transesterification (1-2 mg of lipid) (Christie, 2003).  

If you have 500 ml of plasma, and after the lipid extraction you use 2mg of that lipid to obtain the fatty acids, using a 5% of IS, the formula you would need to use would be like this= ((5 x area of the fatty acid you want to transform x area of your IS)/100) x (amount of lipid that you used to obtain the fatty acids in ug/500). The result would be ug of fatty acid/ul. If you are using tissue, you need to refer to dry weight or protein contents. 

As External standards we use several mixes (RESTEK food FAME, Menhaden oil FAME and  F.A.M.E. Mix, C4-C24), and we inject them twice. At the beginning and at the end of your runs. 

I hope this help!