LC-MS/MS - Change of matrix - problems!!!!

[smartin]

Hi,

I've a doubt and I hope that somebody could help me!!!
I'm developing a LC-MS/MS method for the determination of two molecules in seminal plasma. I'm working from a previous developed and validated method for the same molecules in plasma.

I'm not using a deuterated as IS (I know I should, but for the moment, I can't), and I use the same IS for both molecules. The method for plasma is ok.

For the method in seminal plasma:
What I have obtained is a reductions of the area and also the signal of the IS along the sequence
It doesn't happen for the analytes.

Could somebody give me some explanation about it?
Do you think it could be related to matrix effect?
How can I test it?
Which will be the solution?

I'll wait for all your comments!!

Thank you very much!!!

[LiVD]

Matrix effects are very common with ESI, and can both boost or decrease your signal. The easiest way to cope with this is using matrix matched calibration curves.

[smartin]

Thanks,

I'm using matrix matched calibration curves (in the same matrix)....
do you think that the problem can be still related to matrix effect?

[LiVD]

It might... It all depends on how different your samples are from each other. Are your areas stable if you inject the same sample multiple times?

[smartin]

Hi,

Now I'm trying this. As I think that maybe the area of all the analytes can be afected.
In this case.... maybe problems with the injector????

[smartin]

Hi,

Could somebody help me on this?

Injection of n=20 times the same sample in plasma >>> method validated >>> area constant
Injection of n=20 times the same samples in seminal >>> method not validated >>> area constant
Injection of n=20 times the same sample in plasma >>> method validated >>> area constant

Today:

Injection in seminal:
1.- Calibrators (n=7 with increasing concentration)
2.- Quality controls (4 levels, 5 times each level)
3.- Calibrators (the same as before)

The area of calibrators is ok (not decreasing); then, a decrease of the area in QC, and after, the area of calibrators is increased another time!!!

The calibrators and QC are prepared in the same way: stock solution, and preparation of working solutions (of CAL and QC) that are added, at the same volume, the same volume of blank matrix.

Please, some help??????

[smartin]

Sorry,

Some specifications….
The A of the IS is decreasing along ALL the samples (CAL and QC), and also in the plasma samples processed after the seminal plasma samples…
So, it is clear that is something related with the “new” matrix. Maybe something is getting dirty because of it? Maybe the column or maybe the MS/MS?
Now, I’m going to clean the column, and after that, I’ll reinject some of the same samples. I think that, in this way, if the signal (and area) increases another time, what is going dirty are the column.
Do you agree with me?

In this case…. How can I get with it?

Thank you very much

[LiVD]

That sounds like a reasonable step, although I'd expect your peaks to be tailing if this were the case...

You also might want to check your injection system. Are you sure it always injects the same amount?

[smartin]

Hi,

No, I'm not sure at all....
I should also test it, maybe...

[smartin]

But if after the wash, the area increase again...
it will be strange to be related with injection system.

Isn't it?

[James_Ball]

But if after the wash, the area increase again...
it will be strange to be related with injection system.

Isn't it?

If the wash for the column flows through the injector maybe it will clean both(as in an Agilent autosampler).

[smartin]

At the end, it seems to be related to the MS/MS itself....
So, the new matrix is so much dirty, and it's puting on the tube transfer

[emeulman]

How do you prepare your samples, before you inject them with your system?

[smartin]

It's just a protein precipitation plus evaporation and reconstitution.
I'll try to reconstitute in a higher volume....

I hope it will be better

[emeulman]

Is it possible with your components to analyze them with APCI or will you get no sensitivity?