Dear colleagues,
Currently ,I am making some new molecules by enzymatic catalysis, due to the fact that I do not have standards for these new molecules, I decide to make indirect quantification of the amount of these molecules by determining the amount of the reactants. In order to determine the amount of reactants, i need to make a standard curve for each of them. Because the high cost of the standards, i would like to know if I can make a standard curve with only 10 mg of standard?
Your king answers are highly appreciated
Hi Alkassoumi,
which concentration range do you want to cover and how many points do you want to have on your calibration function? As a rule, a 5-point calibration is sufficient. From an analytical point of view, 10 mg is quite a lot. This should loosely be enough to create a calibration function. I would dissolve the 10 mg and use it as a stock solution from which you can then make a dilution series. But please do not add another dilution from the dilution. I only say: error propagation !
Regards
Markus
Dear Hamadou
It is little tricky to perform indirect quantification of the reactants and your new molecules because of their relevent solubility, polarity, Pharmacochemical property and analytical behaviour.
For this you have to decide relative response factor.
For making analysis easy, you have to go for similar analog for your new compound (with similar physicocheical property).
10mg is sufficient to make stock solution of 1000 ppm. you may go for serial dilution as per the response in LC.
How about your working range? It depend on the working range. Normally do you need at least three points in your standard curve. But better to have more than 3 point.
Thilini Madurangika Jayasinghe I would just to make a standard curve in order to quantify the amount of components in my reaction.
As a example, when we measure the aflatoxin in milk sample, the EU/EC regulation limit is 20 ug/L for B1. hence no need to prepare the standard curve above the 100 ug/L. when we preparing standard curve we need to some points nearly in the lower values and have higher distance between the higher value. If you need to quantify your compound, you may have idea about ''how much you expected or the regulation limits or previous studies results"'. After that you can decide your working range of the standard. And also check your instrument detection limit. Thanks and best wishes for your work
Sorry I have a mistake..The regulation limit of 20ug/L, not in a milk, it is in a feed sample.
Yes,
You can make the samples of different concentration by dilution and make calibration curve. I think four point calibration curve would be sufficient.
Dear Alkassoumi Hassane Hamadou,
To get better idea about about you calibration an possible lowest limit, if you can follow the answers provided in the link below. Also, you can start your calibration curve from Signal to Nise ratio, 3:1, to 10 mg withing the calibration range. If you get levels above the 10 mg for actual samples, you have dilute sample and maintain the elves within 10 mg and back calculate with dilution factor to determine the actual levels in the samples.
thank you.
Piyal
https://www.researchgate.net/post/How_can_I_calculate_the_drug_concentration_from_a_HPLC_analysis_data_report
Yes. But you would have to dilute the 10 mg to 1 L so you have 10 ug/mL and inject only 10 uL (since HPLC is so sensitive). Alternatively, if you have an ultrabalance you can weigh out 1.0 mg (1000 ug) and dilute to 100 mL with mobile phase.
@ A. G. Piyal Aravinna , Buce Neagle Thank you so much for your help
10 mg should be enough for constructing standard curve. As suggested by others, you should make a stock solution in water/acetonitrile or water/methanol and make working stoco from that. You can statt from 10 ug/mL and go by a 2 step or 4 step 7-10 point serial dilution which will give you the idea about the sensitivity and linearity oF the compound in HPLC.
You are welcome. By analyzing the serially diluted samples you will also get the idea about the LLOQ of the method. It's a very standard practice in method development. And please excuse me for the typos in my previous comment.
yes, actually on a previous work we've done a calibration curve with 1 mg of a secondary metabolite in 1 mL of ethanol (1000 ppm), then you should do a series of dilutions ensuring that your unkwoun sample fits in.
ex. 1000 - 15 ppm or less
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