Hi, I think with GCMS or other chromatography systems (HPLC/LC-DAD/MS/MS-MS, TLC/HPTLC-densitometry) different compounds might have same retention times (Rt/Rf), but different Rts must be from different compounds. Different compounds could have different response-detectors , so it means different compound with identical concentrations could showed different peak areas/peak heights. 

For GCMS it is recommended to use SIM (Selected ion Monitoring) method to quantify compound(s), or MRM (multiple reaction monitoring) if you use GC-MSMS. You can make calibration curves for each compounds by using SIM or MRM, than you can estimate the concentration of your target compounds by the calibration curves. Please do not forget to validate your method first, before doing any routine quantification. Good luck

Hello, I don't have specific experience with GC-MS analysis of FAMEs, only GC-FID, but I would suggest that any with protocol you develop you check the response for each compound, especially as unsaturated compounds will interact with the stationary phase slightly differently to the saturated compounds. Assuming that compound "A" behaves the sames as compound "B" always ends up with inaccuracies. If you change column at some point, even if it is the same formulation but a different batch, there is no guarantee your calibration will remain valid.

A simple means of testing would be to dilute your stock 10:0, 16:0, 20:0 mixture by known amounts until you reach the limit of detection for one or more compounds. If one disappears before the others, then it is quite likely the calibrations are not going to be the same.

FAMES fragment to give lots of low mass fragments which are not specific to the particular FAME, so total ion chromatogram response factors are similar for different FAMES.  SIM does not work well for FAMEs. Whether they are similar enough for you to reach the accuracy you need depends on the requirements of your analysis. 

Make up a standard with equal mass fraction or concentration (depending on whether you want to work in Moles or mass) of each FAME and compare peak areas.

Hi, attach 4  application notes for FAME that used SIM  for quantification by GC-MS (2), used full scan (1) and 1 using GCMS MS, good luck!

Two of those notes deal with the specialised and atypical analysis of a restricted suite of  FAMES in a hydrocarbon matrix, which is a completely different analytical challenge to the usual analysis of FAMES from foodstuffs, and the other one; GCMS_5991-1437en-analysis-fatty-aci...-esters-agilent-5975t-ltm-gc-ms.pdf is a full scan method.

In general, for analytes whose spectra lack distinctive fragment ions in high abundance, the tradeoff between detectability and selectivity does not favour SIM. FAMES fall into this category. Compare the spectra for two FAMES at http://webbook.nist.gov/cgi/cbook.cgi?ID=C112390&Mask=200 and http://webbook.nist.gov/cgi/cbook.cgi?ID=C124107&Mask=200 ; the distinctive fragments for each ar at less than 1% of the total ion signal.

Hi, I never work with the two described compounds. What do you think if we used SRM (GC MS MS) for quantification of the 2 compounds?

Dear all,

Thank you very much for all the answers and valuable discussion.

Also thank you for the references Prof. Gunawan.

Peter: I just want to clear something. So for all FAMEs, the area will be same for the same concentration. Is that right?

For example I have a sample with C8:0, C12:0, C14:0, C16:0, C16:1 and C20:0, and I only have one standard compound, say C14:0. Once I've got the area of all the sample, I can use the standard area to calculate the the concentration of other compounds? Or do I need standards for each of the compound?

I actually only need the relative percentage of each FAME in the sample, not the exact concentration. Initially I thought I can use the area to calculate the percentage. So once I get the area of each compound I can calculate the percentage of each compound over the total area. But it seems not correct if the response of the detector is not the same for each compounds.

Hello Safri

The area will be similar for a similar mass of each compound; NB similar but not the same. Maybe they will be similar enough for your purposes, maybe not; it depends on how accurate you need to be.

You will also probably see some inlet discrimination between C8 and C20, so for really accurate work you would need standards of each compound. FAME standards are readily available.

Unless you have interfering peaks from non-FAME compounds an MS offers no advantages over an FID for this analysis. The FID has very nearly equal response factors for all FAMES.

Hi Safri, in addition, for proving the selectivity of the method generally at our laboratory, we did identity and purity check for each of target peaks; and for doing quantification using area sample/area std (single point calibration), please read the recent book of Dr. J. Ermer & Dr.P. Nethercote (2015), Mehod Validation in Pharmaceutical Analysis, VCH-Wiley, p146, Table 5.14 regarding its requirement.

I do agree with Peter Apps and Gunawan Indrayato, but I would complement with a few ideas on numbers: very well calibrated GC-FID systems or MS Systems might reach acuracy or correctnes of a few % absolute. Using good intenral standards, and a calibration function for each compound. However that is not always needed.

If e.g. only a relative change is of interest, e.g. before incubation and over several time points during incubations than, of course it is more importants to be correct relative to the stating point. That can be reached with calibrating the system with just e.g. C 14:0 and inject all samples in the same series. A relative correctness of a few % might be expected, but the real concentrations would be considerably off, especially using GC-MS.

GC-FID can in principle be calibrated as ng carbon on column (ie. independently of the compound) but this requires a chromatorgaphy and an injection system that works free of dicrimination. - I assume for an experience FAME person that can be done, using on column injection and a well maintained column. If all works well, I would expect accuracy of around 10%.

For GC-MS that is by theory not possible. If tried I would expect you hit the right order of magnitude, eventually you can get down to a few 10% but in reality I would not believe you get better than e.g. 30%. - It can get a lot worse, but you have no means to control.

However, sometimes even that is good enough.

Thank you very much for all the valuable discussion and suggestion.. it really help me..

I agree with all suggestions, But as  Peter stated: you need to get the standard for each of the compounds because the response from each compound of FAME will differ even when the concentrations are the same. I use GCMS to quantify FAME never tried using SIM mode -thank you prof Gunawan i will also check the ref you gave , the peak area you get depends on the concentration. Respose may be different for different detectors e.g FID and MS...