If I use C19:0 as an internal standard adding before methanolic HCl transesterification, how to determine the amount of C19:0 ME in the FAMEs for GC analysis? What about using C19:0 ME instead of C19:0?
e.g., add 5 μL of 10 mg/mL C19:0 into 1 mg of the extracted lipids; after transesterification, dissolve the FAMEs in 0.5 mL hexane for further analysis.
Mr. Lin,
Please consider my answer to the following question/discussion and the corresponding reference section, therein:
https://www.researchgate.net/post/Which_type_of_sample_is_preferable_to_identify_chemical_compound_from_plant_sources?_ec=topicPostOverviewQuestionRecommendations&_sg=x_BA_7dNOphg9ZvkPlcO1DTmsA3rcN1cGre2cmzCEuwwLSt8LCl6wcZD4ohOMuC53bESkFNO8RclrBBr
If you assume that transesterification undergoes with same effectiveness for all fatyy acids you don't need to know this info. You simply relate the content of IS and its area to the analytes of interest. If you need it to do more accurately you need to determine the recovery.
If you are using FID you can compare the peak areas of C19:0 and its Me ester to estimate the rate of transesterification
Using area ratio for FID is quite accurate (still if you need to make it more accurate you need to know responce factors or have a standard of Me-C19 to determine it).
Regards
GB
@Bojidarka B. Ivanova your answer is not helpful (the provided as well)
It's usual to use an FA with an odd number of carbons as internal standard for this analysis (often C19:0 or C17:0). Depending on accuracy requirements, you might use FID response factors instead. However, if known instrumental and other sources of bias (for example due to the injector) are considered significant, you will have to validate and calibrate ("on the day") for each target analyte.
If your specimens might contain odd-carbon FAs, you will need to use either external standardisation (which can be reasonably a precise and accurate with an automatic injector), or standard additions (perhaps only one or two additions for a method that has been validated by means of a full calibration curve).
If the putative odd-carbon acids are minor constituents, I would question the specificity (selectivity) of a method with FID. The metabolomics literature provides reminders of the number of natural products and artefacts that may be encountered. Here, it may be necessary to use GC-MS, which provides a spectral fingerprint. The estimated selectivity is also impoved by using an isotopic internal standard, because the retention time is measured with less uncertainty than with an external or homologous one.
Finally, I don't like getting involved in polemics, but consider from experience that it would be better to abandon a project that to use analytical methods of unproven selectivity. Recently, I carried out an extensive review of a three decades-long controversy that arose and persists because of the persistent use of inadequate and unvalidated analytical methodology, together with what appears to be widespread failure of the peer review system in this respect:
DOI: 10.13140/RG.2.2.10144.53765
Hi
Yes. you can do the addition before transesterification, but you need to know how much exactly you added. So later when you have analysed your samples you can subtract the internal standard conc. As the following paper
https://doi.org/10.1007/s12161-021-02203-0
I hope this helps!
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