It is difficult to make any suggestions without a full description of your sample, fatty acid extraction and derivatisation, GC/MS parameters (temperatures, column) and a copy of the final chromatogram.

We are using 5sil ms coloum with temperature gradient 60-280 @15 degree Celsius ramping. We were developing a new derivatisation method. All the peaks are resolving good and we have also done the mrm optimization in accordance. The problem is with the part on linearity for quantitative estimations. Ideally the method should be standard addition to negate Matrix effects for example fecal samples. But with this we have challenge of generating linearity with every batch of samples. To over come this we can use internal standard correction. But doing this not possible in the software either we can have standard addition or internal standard.

If as you seem to be saying, you have achieved baseline resolution of all your peaks and you have a deuterated internal standard, then I fail to understand your problem. Quantify against the internal standard. Standard addition of all your peaks for all your samples would represent a lot of additional work, for little or no gain. If the Shimadzu software is a problem then you should be able to plug your peak areas into a spreadsheet for your calculations. alternatively there may be some freeware you can access.

It has been a while since I used Shimadzu software, but I do recall it has internal standard calibration curves capabilities. Here is a reference which should be helpful: https://www.shimadzu.com/an/sites/shimadzu.com.an/files/pim/pim_document_file/applications/application_note/13049/m220.pdf

During my toxicology contract testing lab days, we used synthetic biological matrices for developing our methods. What stage are you spiking your standards? Post or pre liquid-liquid or solid phase extraction? We found that the key critical steps were dativization recovery and reproducible extraction. I would also check to see if you can export your data out of the Shimadzu software as a comma or tab delineated file and import it into Excel as recommended by Charles Hocart. This will reduce transcription error, and could be the fastest way to continue moving forward.

Michael Sanford We are doing spiking in pre liquid-liquid phase extraction. The current software of shimadzu is restricting user only to use either internal standard method; where linearity is drawn concentration ratio vs response ratio. Or standard addition, where reverse interpolation of x axis is being done. there is no way possible to do internal standard normalization, where we can calculate linearity between concentration with response ratio and do reverse interpolation. Even the method you have shared is doing the same thing i am referring to but unfortunately it is not possible in software.