Depending on the information you want to obtain, internal normalization and internal standardization would give appropriate results. If you need to now the proportions of fatty acids (on a weight or molar basis), internal normalization would do it. For instance if you need to know the actual amounts of fatty acids in the sample, you will need to use one (or more) internal standard (usually odd-chain fatty acids). For example, if you need to follow (bio)-chemical transformations taking place in a soil sample, it is better if you use internal standardization. External standardization is not advisable using conventional split/splitless injectors.

Thank you for your answer. I need to know the relative fatty acid composition of biological samples, with FA values expressed as percentage of total FAs. I understood that I need an internal standard to correct the areas, due to the sample loss into the GC. Then, I do not understand if I need a standard mixture of all the possible FAs to built one o more calibration curve to calculate the final concentrations, or not.

Hi Geraldina. Yes you will need a set of standard mixtures with a fixed amount of internal standard in each, but different FAME concentrations. A minimum of 4 samples each with different FAME concentrations would suffice if you have the equipment to accurately measure the samples.

The Internal Standard is not really doing anything about sample loss inside the GC, rather the difference in Respose Factor (RF) for each of the analytes. Hence it is important that your Internal Standards are quite similar in size, polarity, structure to your target analyte. You may need a number of Internal standards if you are looking at a broad spectrum of FAs.

Thank you Mark. The unique problem now will be find quantitative standards of all the possible FAs in biological samples i.e. around 70 or even more! Most of them are sold as a qualitative standards or are not for sale at all. We've bought Supelco® 37 Component FAME Mix, and the orthers? How can I quantify them if I can't build a calibration curve for them? Further, if I'll find them all, should I mix them all together to built one single calibration curve at different concentrations of FAMEs or I can do different mixtures and apply them one by one to my unknown sample?

Hi, it would be interesting which detection system are available in your lab. If you are using MS as detection system you will need full FAME sets to do the calibration work, since unique fragments of different FAME species will give different signal intensity. A practical solution for quantification of different FA species is detection via FID. Thereby signal intensity is independent of FAME species. For high abundant mammalian FAs i can recommend Larodan fine chemicals. They have some useful sets of FAMEs. The addition of internal standards (FAME C17:0 C19:0) to your sample gives you reproducible information about the loss of analytes during extraction and isolation. Good Luck

Thank you very much Thomas. I've found all FAMEs I was searching in the Larodan web site and I've already aked for a quotation! Wow! In the lab we have both GCMS and GC with FID. I've used GCMS for some previous analysis but now I would like to use FID. In addition to use internal standards to get information about the loss of analytes during extraction and isolation, means like mixing surrogates, do you think it could be useful putting an internal standard at the end of the extracion process to get information about the response only of GC/MS or GC/FID? Last question: would you suggest to me to analyse also certified reference materials for validation of extraction methods?

Hi Again - looks like you have got some great advice here!

Regarding your last question, you don't really need to add an internal standard (I.S). again after extraction. By preparing your calibration standards with I.S. and spikes of each FAME (you can add multiple analytes to the one calibration standard), the software will calculate a Response Factor for each analyte. In other words, once you have calibrated for each individual analyte, you no longer need to consider the response factor of your instrument/analyte. That is the advantage of IS quantitation.

Just to confirm, your I.S. needs to be something other than your analytes, but very closely related, otherwise your extraction process will indeed change the relative concentrations of standard to analyte. The best I.S.s are isotopic modifications of the same molecule (Eg acetone's best I.S. would be acetone-d6). If your IS is too dissimilar, then your error will start to get bigger.

Hi. I am glad that I can help. Concerning internal standards. If you measure your samples with GC/MS the best but also the more expensive option is, like Mark Bali wrote, stable isotope labeled FAMEs. In case of GC/FID this is problematic since there is no possibility to differentiate between non-labeled and e.g. C13-labeled FAMEs using FID detection.

In your case i would suggest to add C17 or C19 FAME or both directly to your sample prior to extraction and further processing. This will give you a robust information about the specific loss of material during FAME preparation. Odd chain FAs can´t be found in mammalian samples and will be nicely seperated from even chain FAMEs. I also think (see Mark) that there is no need for an addition of I.S. before GC injection.

Of course, validation is always a good idea. You can do this by directly measuring a certain amount, lets say xx nmol of your supelco FAME mix (which is more or less a certified mix). Subsequently you use the xx nmol and perform the whole extraction, isolation and transesterification procedure. A comparison of these results tells you the quantity of your extraction procedure and also if all FAME species are effected simmilarly.

If you decide to use GC/FID it is also important to check if the response of the FID is stable. Just make calibration of different single FAMEs (e.g. C14:0, C16:1, C18:3....) to see if you receive the same signal/concentration for saturated, monounsaturated and polyunsaturated FA species.

Again....good luck

Sorry Geraldina...my suggestion to validate your extraction with the FAME Mix was not the "best". FAMEs will have different extraction properties as compared to free fatty acids (FA). So it will be better to use a FA mixture. Just compare the FID signals of an FA mix sample, which is transesterified, and the same amount of the FA mix, which undergoes extraction and transesterification. This should work better.

Thomas and Mark, many thanks to both of you. You gave me very precious advises. I'm sorry to bore you but there is still somethings eluding me about I.S. We have bought single odd chain FA and FAME so it could be better to use them instead to buy more things. Does the same I.S. is useful at the same time to give information about the loss of analytes during extraction and isolation and to let the software calculates a Response Factor in the calibration procedure? Means: should I add an odd chain FA at the beginning of the sample extraction to verify the extraction and transesterification procedure and should I add the respective odd chain FAME in the calibration mixture?

Hi, Geraldina. Just one comment about the type of IS you can use: I would recommend you to use 15:0,17:0 or even 23:0. The problem with 19:0 if you are using GC-FID is that this FA can overlap with gamma-linolenic acid (GLA, 18:3n-6), and this can be a problem if this fatty acid exists in your samples.

Even with a very polar column specific for FAME like the BPX70 (25M X 0.22mm, 0.25um; http://www.sge.com/products/columns/gc-columns/bpx70) ?

Hi Geraldina. If your aim is to know the exact concentration of your target analytes before extraction, then each of your calibration standards must go through the same extraction process, otherwise it remains unknown what your work-up losses are. For example

-Prepare 5 calibration samples of target analytes at precisely known concentrations, which span the expected concentration range of your unknown samples. Duplicates of each sample are often recommended to reduce error.

-Make a stock solution containing your Internal Standard/s (1 or more) in preferably the same solvent. The concentration of your stock will be determined by the size of your samples (for example I use a 0.2M stock of Ethyl Acetate IS so that I can add a 20uL spike to each 2mL sample, making a final concentration of 2mM.) The final concentration should be around mid-range of your expected analyte concentration range.

- Add your carefully measured aliquot of IS stock to each of the calibration samples, then make up to the mark to ensure accurate concentrations.

- Conduct your full extraction procedure (and derivitization) on each sample and run on your machine. Running a blank between each calibration sample is a good idea.

In this way, the exact extraction loss for each relative concentration is able to be identified. Large scatter or error means that the extraction procedure needs to be tightened up, or your IS is not similar enough to the analytes. If you can't reproducibly quantitate the concentration of your known standard solutions, there is no way to produce accurate concentrations for the unknowns.

I think there is little point in adding another IS after extraction , as even in the calibration standards the concentrations at this stage are to some extent unknown. There may be some way to utilise the second IS to mitigate injection error (a sort of double internal standard), but I haven't come across this before - I would be interested to know if there is such a process!

Hope this helps.

The column is only 25 m long? I think it is such a short column to get a good resolution of certain fatty acids, specially to separate cis and trans isomers. But I've never used that column before, so can't tell you about resolution problems with it. Anyway, I am curious about the results that can be obtained with that normal-phase column...

Mark your answer is very useful. What you have written is exactly what I was planning to do. Thank you very much.

Miguel this column is:

Custom designed for separation of Fatty Acid Methyl Esters FAMEs

Ideal for cis/trans isomer separation

Then I hope it will be the best for my aims. I will let you know when I will start to use it! It is just bought.. Further I've made a mistake. It is 30m long not 25. It is not markedly different but I hope it will works well!

Sorry for the delay! I can only fully agree with Marks answer. To do it like this should work well!!!

Good luck

Yes, thank you, Geraldina. I am really interested in the results that you can achieve with that column, so would be nice if you let me know! Best regards.

Thank you everybody! I will let you know how my work will go on!

Dear Geraldina,

I am a Senior Chemist at SGS Canada Inc and have been doing fatty acids profile by GC-FID for many years. I use the AOAC 996.06 official method as reference, being part of AOAC, AOCS check saple program and my results are well within acceptalbe range. I use C11:0 -undecanoic methyl ester as internal standard for my FAMEs mixed standard solution, C11:0 triundecanoin as internal standard for my actual sample. My GC column is SP 2560, 100mx0.25mm with 0.20 um film and best column for GC Fat analysis, I am able to separate around 45 differant fatty acids in 60 minutes. LOQ can go down to 0.01 g/100 g.

Regards,

Kaiser

Kaiser Turabek,  I could use your help.  I am using the same column to analyze FAME.  My results are coming up more heavily weighted toward polyunsaturate fat particularly 18:3.  I am under-estimating saturated and monounsaturated but over-estimating 18:2 and 18:3.