In order to measure phytosterol concentration of O/W emulsion, I have used single point internal standard method i.e. calculating the response factor, using areas of known amount of IS and the compound of interest. Then adding a known amount of the IS to the sample containing analyte of unknown concentration and finally calculating the analyte amount using respective equation. Could you please let me know does this method require recovery assay? Or it is not applicable due to the usage of internal standard? Thank you in advance.
Hi, I think beta sitosterol can be determined quantitatively easily by using GC (FID/MS) without any derivatization. but if you want you can do also deriivatization (e.g. with sillyI), according our experiences no need to do derivatization for analyzing sterols. You can use semipolar columns such as SE-30 or HP-5 or others. Sitosterol will be separated with stigmasterol, campesterol, cholesterol or other sterols. For doing any analysis I think it is nice if you do validation method first (selectivity, accuracy, precision etc).. You can do with external or internal calibration to do quantification.,
https://pubag.nal.usda.gov/pubag/downloadPDF.xhtml?id=24592&content=PDF
http://agriculturejournals.cz/publicFiles/07775.pdf
Thank you so much for your answers
I used GC/FID method but no peak was detected without derivatization. So, the sample was derivaitized using BSTFA containing 1% Trimethylchlorosilane.
5 a cholestan was used as an internal standard and added to the sample before saponification so it will account for any change or loss during the process. however, I wonder why the recovery of the method is around 70%?
considering this situation, should the measured concentrations of samples multiplied by 100/70 to estimate the actual amount of sitosterol present in sample?
would you please let me know the exact procedure of quantifying sterols without derivatization?
thank you for your consideration and time.
Hi, Can you describe what kind of sterols in your sample? How were many peaks of sylil sterols in your sample? Do you have pure standards sterols e.g. Sitosterol, Stigmaseterol, campesterol, Cholesterol, brasicasterol or other sterols? I think sitosterol, stigmasterol and others must be appeared without any derivatization. If the recovery ca 70 %, I think you should evaluate your extraction methods. In my opinion is it not nice multiply the results by 100/70, best regards
Hi
Thank you again.
my samples contain beta sitosterol that I have added the known amount of it to them. The compound is beta sitosterol and the impurities are beta sitosatanol and campsterols according to manufacture report. the obtained peaks are very sharp and distinct and we can identify them very easily. now I am doing some preliminary tests to optimize the method. probably there is some problems with the extraction method that causes sample loss. I appreciate any other suggestion to increase the accuracy of method.
Best regards
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