Bioequivalence and Bioavailability Forum

Dear Auditor,

❝ In other words if the plasma volume is increased 1.o ml from 0.4 ml then is it true that we can achieve more sensitive LLOQ?

It depends on the method you are using.

If you are using protein precipitation, with no evaporation: it will make no change. You will keep the same ratio of plasma to precipitation reagent (e.g. acetonitrile). Let's say you precipitate your 0.4 ml of plasma with 0.6 ml of ACN and inject 20 µl of the supernatant: you will now precipitate 1 ml of plasma with 1.5 ml of ACN and still inject 20 µl. No difference.

If you are using solid phase extraction (SPE), and keep the same elution volume (e.g. 200 µl), or liquid/liquid extraction (LLE) with an evaporation step in the end and a reconstitution with an unchanged volume, you will indeed obtain a more concentrated extract and it may improve your LLOQ. But remember that you will also more concentrate possible interfering substances, resulting in a higher background noise, and possibly more matrix effects. You may also have some problems with recovery if you saturate your SPE column or don't increase your LLE solvent volume.

So even with SPE and LLE, there is no constant and strictly linear relationship between the plasma volume and your LLOQ. You need to try and see what happens in your own situation.

Remember also that increasing your sample volume may require to increase the blood volume collected from the trial subjects. It will also increase your needs for blank plasma to prepare CC/QC samples. And if you have already validated your method with 0.4 ml, you will have to do a full revalidation with 1.0 ml (except stability in matrix).

Regards
Ohlbe