let's say I injected a plant extract (5mg/ml) into a HPLC system (1200 Agilent series) with injection volume of 10ul. So technically, I am introducing 0.05mg of extract into the HPLC system and get my separation. At the end of the run, the software (chemstation) will process the data and integrate the area under the graph (AUG) of all detected peaks. Let's say that I know the compounds from the extract and their retention time (have been identified previously via LCMS-MS), instead of using a standard (purchase from vendor), do serial dilution, make a standard curve and quantify as per usual, can I use the AUG value to relatively quantify the compound? For eg, (AUG of a particular peak / total area count of all peaks) X 0.05mg will give me a relative mass of that particular compound as in 0.05mg injection. Has anyone done that before and published your result in any journals and get accepted? I wonder that :-) Thanks
Nope!
1. Each molecule or peak has its own response factor for that particular detector. Thus peaks can have the same size (AUG) but have a radically different mass.
2. Peaks from the placebo that are not your molecule of interest will appear (similar to the injection or solvent front) and can radically alter mathematically the AUG result you obtain and publish.
Obtain a 'pure' standard (99.0%) and perform the 'analytical method validation' as required by ICH Q2(r1). Your 'reputation' relies upon doing 'good' and 'competent' work!
1. If you have LC-MS why would you drop back to a semi-qualitative technique?
2. Do you have quantification data from the LC-MS? If so, use that sample as a quantified standard. If not, you need a standard.
3. It isn't published in journals because, as Bruce points out, it isn't valid and no peer review process would ever let it through.
All instrumental analyses are relative methods with few exceptions i.e. the absolute value of a signal has no meaning until and unless you have a response from the standards as well. In the same way, the area under a peak, has no "absolute" meaning. It is proportional to concentrations, but where is the proportionality constant. Please don't do calculations the way you suggested in the original post.
Thank you so much Bruce Neagle, Mark Krause and Farooq Wahab. Your answer clarified my doubts. Really appreciate it guys :-)
You can use LC/MS/MS to identify a peak but you will need an organic chemist/lab to manufacture a reference standard. The standard then has ti be 'identified and characterized' by NMR, UV, FTIR, MS...
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