Ladi
☆    

Thailand,
2015-09-17 09:04
(3116 d 06:51 ago)

Posting: # 15413
Views: 7,870
 

 spiked samples preparation [Bioanalytics]

Dear all BA/BE people,

This is my first time posting here. I am really glad to find this forum.

Would you please elucidate me the correct and most appropriate way to prepare spiked plasma samples for calibration curve and QC. First let me tell you how we usually do it.

For example, if we need 500 uL of study sample - we would use 450 or 480 uL of the blank plasma and add 50 uL or 20 uL of analyte solution (working solution) to make total volume of 500 uL (So that concentration is calculated from total volume of 500 uL). Our analyte solution is typically prepared in 50% methanol or acetonitrile. I understand that doing this way we have diluted the matrix in spiked samples and it won't be equivalent to study samples.

What I understand we should have done is actually used the same volume of blank plasma and study samples. For example, we could use 500 uL of blank plasma and then spike with 20 uL or 50 uL of analyte solution. Then, what we should do is add 20 uL or 50 uL of diluent to all study samples too.

Also I understand that the %solution spiked into plasma should not be more than 5% (not sure where this number is from) of total plasma volume. What i am not clear is 5% 'organic solvent' or 5% 'spiking solution'?

I really appreciate your kind suggestions,

Ladi
Ohlbe
★★★

France,
2015-09-17 12:45
(3116 d 03:10 ago)

@ Ladi
Posting: # 15416
Views: 6,665
 

 Spiking volume

Dear Ladi,

❝ This is my first time posting here.


Welcome !

❝ What I understand we should have done is actually used the same volume of blank plasma and study samples. For example, we could use 500 uL of blank plasma and then spike with 20 uL or 50 uL of analyte solution. Then, what we should do is add 20 uL or 50 uL of diluent to all study samples too.


That would indeed be the best practice. Especially with a large spiking volume. Unfortunately, this practice is not that common.

❝ Also I understand that the %solution spiked into plasma should not be more than 5% (not sure where this number is from) [...]



Pure rule of thumb, AFAIK.

❝ [...] of total plasma volume. What i am not clear is 5% 'organic solvent' or 5% 'spiking solution'?


More than 5 % organic solvent is likely to denaturate proteins to some extent and can affect protein binding and recovery. More than 5 % spiking solution will dilute the sample, which may change recovery and matrix effects. If you add the same volume of diluent to all samples you will get the same effect in all your samples. But due to the possible combined effect of freeze/thaw I would still avoid higher spiking volumes.

Regards
Ohlbe
Ladi
☆    

Thailand,
2015-09-17 15:02
(3116 d 00:53 ago)

@ Ohlbe
Posting: # 15418
Views: 6,627
 

 Spiking volume

Dear Ohlbe,

Thank you for your response. You have cleared my doubts.

The lowest spiking volume we ever use is 20 uL. Lower than that would not be practical for our lab. That would limit the plasma volume I can use too i.e. cannot go lower than 500 uL (20 uL of 500 uL is 4% already).

Thank you,
Ladi
Ladi
☆    

Thailand,
2015-09-30 15:42
(3103 d 00:13 ago)

@ Ohlbe
Posting: # 15498
Views: 6,018
 

 Spiking volume-total volume

Dear Ohlbe and other members,

Sorry to come back to this issue again but people at our lab are having problem with this idea (including our beloved QA).

❝ [...] we could use 500 uL of blank plasma and then spike with 20 uL or 50 uL of analyte solution. Then, what we should do is add 20 uL or 50 uL of diluent to all study samples too.


In my latest method SOP I stated that:
  • Cal curve, QC are prepared from 480 uL blank plasma and 20 uL spiking solution (in 50% methanol) before extraction.

  • To analyze study sample pipette 480 uL of the study sample and add 20 uL of 50% methanol before extraction.
The question people are having is should the drug concentration be calculated base on total volume of 480 uL or 500 uL?

I hope this question is not too silly,
Ladi
ElMaestro
★★★

Denmark,
2015-09-30 15:57
(3102 d 23:58 ago)

(edited by ElMaestro on 2015-10-01 00:19)
@ Ladi
Posting: # 15499
Views: 6,070
 

 Spiking volume-total volume

Hi Ladi,

❝   Cal curve, QC are prepared from 480 uL blank plasma and 20 uL spiking solution (in 50% methanol) before extraction.


❝ – To analyze study sample pipette 480 uL of the study sample and add 20 uL of 50% methanol before extraction.


❝ The question people are having is should the drug concentration be calculated base on total volume of 480 uL or 500 uL?


I don't think it is a silly question at all. I bet most of us make mistakes and are in doubt very often when we calculate dilutions and concentrations. It is a serious matter. My own chemistry teacher had to take early retirement because she was so perplexed by my persistent miscalculations.


Late correction - I seem to have goofed it up as usual (yeah, big surprise :no:).

The way I understand you:
You have xyz ng analyte per 20 uL; you then dilute that entire volume to 500 ul before execution. Now your working conc. is 1/25 of the initial concentration.
And you do not have a vial containing anything that is 1/24 of the initial concentration as would be the case if you had to use 480 uL as the final volume.

Pass or fail!
ElMaestro
Ladi
☆    

Thailand,
2015-09-30 16:55
(3102 d 23:00 ago)

@ ElMaestro
Posting: # 15501
Views: 5,982
 

 Spiking volume-total volume

Dear ElMaestro,

❝ My own chemistry teacher had to take early retirement because she was so perplexed by my persistent miscalculations.


:lol2: :lol2: :lol2:

❝ You have xyz ng analyte per 20 ul; you then dilute that entire volume to 500 ul before execution. Now your conc. is xyz/25 units ng/ul (or xyz/(500/20)).

❝ And you do at this point not possess a solution which is xyz/(480/20).


I completely agree with you.

But people are really questioning this because the volume of 'study sample' use is only 480 uL. So they are curious if the total plasma used put in report should be 480 uL or 500 uL. And I am trying to figure out how to explain it.

Still arguing in the lab :no:,
Ladi
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