Chromatography Forum

Hi,

Taking into account the following precipitation method for a LC-MS/MS analysis:

100µl sample + 500µl acetonitril
Vortex and centrifuge
To decant the supernatant into another tube
To add 200µl of formic acid

How will be the matrix effect calculated?
Is it as follows?

Biological samples:
To do the extraction, and at the end to add the analyte (for example, 3µl of solution 10µg/mL)

For standard solution:
To 600µl of water, add 200µl of formic acid and add the analyte (for example, 3µl of solution 10µg/mL)

Is it correct??????

Are you trying to evaluate the matrix effect on the sample prep or on the MS ?

If you are looking at the effect on the MS *only*, then what you propose is fine. If you want to check the effect of recovery through the sample workup, then you need to add the analytes *before* you do the precipitation (this assumes, that you have a matrix blank available and that you don't have to deal with things like conjugates of your analytes).

Thanks at all!!

Yes, I'm looking at the effect on the MS "only". As the method described I think is not really an "extraction" method (it is only a precipitation), I think that it's not essential to calculate the "recovery" itself, but it is to do the "matrix effect".
Is it correct?

However, my main doubts are regarding the calculations of what I do. It means that, being my method ok for lineality, accuracy, precision, LDe and LQe, when I try to assay the "matrix effect" I get very, very; very bad results!!!!
Here it is:

Biological samples >> Areas:
X21: 1088072056; X22: 1151161809; X23: 1368775301; X24: 1421135153
X41: 216241067; X42: 218177960; X43: 219363335; X44: 241196952
Standard solutions >> Areas:
B21: 48548217; B22: 75786619; B23: 25342550; B24: 20292037
B41: 2205097; B42: 1627063; B43: 2031404; B44: 1911837

If you calculate the "matrix effect" as Area biological sample/Area standard solution * 100, you'll see values around 2500% or even more!!!!
I think that it can't be done by an existing "matrix effect", or qm I wrong????

So, I think that maybe there is an error in the volumes I read in the post before.

I'm really, really confused!!!!

Assuming a negligible loss when you transfer the supernatant, your volumes are consistent. So, it looks like the matrix is increasing your response.

Two possibilities for that problem:
- the acetonitrile (easy to check; add your standards to an ACN/water solution)
- the matrix itself. On that score, I can't help (I'm *not* a mass spectrometrist), but some questions come to mind that may allow others to make useful suggestions:
- how are you ionizing (ESI or APCI?)
- are the 21 . . . 44 numbers different compounds? different daughter ions of the same parent?

Hi!!!

So,

The acetonitrile (easy to check; add your standards to an ACN/water solution)
What acetonitrile are you talking about? The acetonitrile used for the precipitation?
-the matrix itself. On that score, I can't help (I'm *not* a mass spectrometrist), but some questions come to mind that may allow others to make useful suggestions:
- how are you ionizing (ESI or APCI?)The ionizing is ESI
- are the 21 . . . 44 numbers different compounds? different daughter ions of the same parent?
Oh no!!! In fact, the 21... 44 are only the numbers of the concentration and replicates: 21-24 for the 2nd concentration (21 the first replicate,....24 the fourth replicate), and 41-44 for the 4th concentration (41 the first replicate.... 44 the fourth repliate)

But could be possible to have so a huge matrix effect with a good lineality, LDe, LQe, accuacy......?????
I'm really, really confused!!!

Maybe I can share it in the LC/MS-MS forum?

What acetonitrile are you talking about? The acetonitrile used for the precipitation?

Yes

good lineality, LDe, LQe, accuacy......?????

I'm missing something here. If those are replicates, you're looking at RSDs on the order of 50% for B2x and 10% for B4x

What acetonitrile are you talking about? The acetonitrile used for the precipitation?

Yes
I don't understand this way to solve it.... Only to the standards? And what about the biological samples?

good lineality, LDe, LQe, accuacy......?????

I'm missing something here. If those are replicates, you're looking at RSDs on the order of 50% for B2x and 10% for B4x

No!!! I've good results with other samples, only biological samples!!!

Your solvents are different. Shouldn't your standard be 100 ul of water + 500 ul of Acn?

And you need to be exact with your sample volumes too. Transfer 600 ul of supernatent.

Thanks!!!

It's true!!! But I added 500µl of water in order to make a standard solution.
In fact, from a modification of the aboved method:

For biological samples: 100µl sample + 300µl ACN >>> precipitation >>> Take 200µl and + 100µl water solution
So, for standard:
200µl of ACN + 100µl of water solution????

But in fact, why 200µl of ACN??
I want to test matrix effects on standard solution, and in fact the ACN is used for the extraction.....

Anyway, I'll try it again with these changes.
I hope everything is going ok!!

Tell me!
And I'll tell you the results, of course!!!

Thank you very much of both!!

Finally it was only a problem regarding the solvents!!!! I had to do it using ACN!
Now it's ok!!
So, I can going on!!

Thank you very much!!!