The amount of internal standard to be added to the sample for analysis is suppose to be about 0.3 to 0.5 of the expected amount of the analyte. How do you determine the expected amount of the analyte? For instance if you are doing a pharmacokinetics studies and 1mg of drug is administered. How do you determine the expected amount of the drug in plasma to help with internal standard amount calculation?
It is not possible to know, you have to make an educated guess. Try to find som literature of similar studies and make a guess out of them in what concentration range the analyte will be. It its not crucial that the internal standard concentration is 0.3 - 0.5 of the analyte concentration. Since the concentration may vary a a lot in a set of samples, it is not possible to fulfill that criteria. Try to find a suitable concentration that is not too low nor too high. If it is too low you risk that it will be close to LOQ in some samples if the recovery is lower than expected or their is suppression of the signal. If it is too high you get memory (carry-over) effects and other problems. It depends on your instrumentation (detector).
It is not possible to know, you have to make an educated guess. Try to find som literature of similar studies and make a guess out of them in what concentration range the analyte will be. It its not crucial that the internal standard concentration is 0.3 - 0.5 of the analyte concentration. Since the concentration may vary a a lot in a set of samples, it is not possible to fulfill that criteria. Try to find a suitable concentration that is not too low nor too high. If it is too low you risk that it will be close to LOQ in some samples if the recovery is lower than expected or their is suppression of the signal. If it is too high you get memory (carry-over) effects and other problems. It depends on your instrumentation (detector).
Use internal standard concentration that gives you similar signal to the reference standard.
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