spiked plasma preparation

Discussions about sample preparation: extraction, cleanup, derivatization, etc.

2 posts Page 1 of 1
Dear Members,

I am doing BE study in plasma. I use LCMS/MS to analyze samples. I am having trouble settling some idea out on mimicking 'spiked plasma samples' (calibration curve, QC) with the 'study samples'.

I construct a calibration curve and their concentration were calculated based on total plasma volume of 500 uL.

In practice, I mixed 480 uL of blank plasma with 20 uL (lowest I can do) of the analyte solution (in 50% methanol). I then extract these calibration standards along with 500 uL of study sample. So in my report, I say I used 500 uL of plasma and get following concentration xxx, xxx, xxx etc.

However, doing this way my matrix is not the same (480 uL blank plasma compare to 500 uL study sample plasma)

So I decided to change it and could think of two options;

1) mix 500 uL of study sample with 20 uL of 50% methanol and extract it against 520 uL of spiked plasma (500 uL blank plasma mixed with 20 uL analyte solution)

2) mix 480 uL of study sample with 20 uL of 50% methanol and extract it against 500 uL of spiked plasma (480 uL blank plasma mixed with 20 uL analyte solution)

now in both method calibration curve and study samples have the 'same matrix'

But,

in method (1), I use 500 uL study sample, but spiked sample is 520 uL

in method (2), I use 480 uL study sample, but spiked sample is 500 uL

Which one would be is more accurate if I want to report my concentration based on total volume of 500 uL??

Please share your idea, I would greatly appreciate it.

Best Regards,
Pratheep
Dear Pratheep,

I would suggest you to go with a bulk spiking i.e spike your calibration and QC samples in more volume above 500 ul
may be upto 1-5 ml in plasma. And now you aliquot 500 Ul spiked CC and QC which in a way similar to your sample aliquoting volume.

Dr.Nagesh
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