Chromatography Forum

Hello everybody,
I am going to use a HPLC method to quantify a compound in liquid fungal growth medium. The method includes SPE clean up. The method uses an external standard for the quantification.

Is it necessary to prepare the standard in the same medium to match the matrix?
Does the standard have to be treated exactly as any other sample including SPE clean up?

Thank you in advance

Not necessary, so long as you can demonstrate 100% recovery during your validation.

Thanks Tom

What if the recovery was less than 100%?

Depends on how much less. If the difference is not statistically significant, then don't worry about it.

If the recovery is consistent, then you just factor that in to the calculation and get on with your life.

If the recovery varies from one day to the next, then yes, you need to start exploring matrix effects.

My general rule has always been that the more closely the standards match the samples, the better the results.

But I have seen some people (published) add a standard before workup for all their work, so they never noticed that they lost as much as 80% of the standard and unknown, giving them "~100% yield". That´s one reason for having suggested, in the past, to use both internal and external standards (without matrix, etc.), concurrently.
In my opinion one has to decide whether one does the analysis to find out what one has, or to satisfy regulating authorities.

Thanks a lot Tom and HW Mueller.

The recovery is 90% +/- 0.82% , so it seems that using a factor in the calculations will be usful.