Internal standard problem:(

[smkh]

Hi everyone
I'm doing now an analysis for captopril in human plasma ,
what i observe now that internal standard in the volunteer samples less than that of calibrators by factor of 10 , means its area in calibration standards and QC's : 50000 but in the volunteer samples 5000 and this appear in all analyzed volunteers with pass Qc's in between the volunteer samples because it is kept the intensity of the standard .. is this called ion supression, matrix effect , what cause it and how to minmize it please
(internal standard is lisinopril)

[sam.pedraglio]

Is there any difference between the anticoagulant in the plasma you use for the calibration/QC and the one in the real samples?
And what about the vehicle used in the treatment of the volunteers?

[lmh]

not sure I fully understand what you've done, but there are two reasons why an internal standard can be low: (1) you lost material during extraction between adding internal standard and the final analysis; (2) you have ion suppression or similar loss of detector efficiency.

If there is any chance that you have lost material, it's worth establishing which problem, (1) or (2) has happened, as they have different solutions.

One way is to take a prepared extract (with low signal of standard) and run it again with and without addition of a further small amount of standard (internal or the analyte itself, it doesn't really matter). Then look to see if the peak area increase on adding material is what you would expect from pure sample. If it is, then you have lost material during extraction. If it is similarly only 10% of what you would expect, then you have ion suppression.

[smkh]

what I see that I have ion supression
and what I want to know more about ion supression and what cause it and how to reduce it please?

[lmh]

depends on what is in the sample. For instance, lots of perhalogenated acids (TFA, TCA) can cause substantial suppression.

[sam.pedraglio]

Ion suppression during biological analyses is normal; ususally it's called matrix effect.
It could be possible that the plasma coming from your volunteers is different from your blank plasma used for the calibration and the qc.
There're only two ways to avoid it:
- change the extraction procedure
- change the chromatography
both of them are needed to separate the matrix components from your analyte (either captopril or IS).

[smkh]

what Itry to do now is changing internal standard with other one have less matrix effect
but how to avoid somthing like this in future, this problem appear when i do succesful validation and start analysis and no supression occur when i change the blank plasma source ,this happened only with volunteers samples!!

[shahis77]

I am also facing the same problem but in my case the response of the internal standard is much higher in volunteer samples than the standards.If this is due to matrix effect it is very difficult to get blank matrix always for method validation and development before the clinical trial samples arrive for analysis.Also we are getting samples from different clinical trials and no body provides blank plasma for calibration standards....so i think this problem can not be solved if it is because of matrix effect...except you have the same matrix for volunteer samples and standards.

[Uwe Neue]

The best way is to eliminate matrix interferences, rather than looking for other internal standards. There are well established methods around for doing solid-phase extraction to eliminate a very large fraction of the interferences. Methods that work in 95% of the cases have been established. Look for the Oasis technical information at the Waters website.

[smkh]

Can anyone please give me agood links to help me in understanding ion supression or matrix effect and the mechanism of its happening in LC-MS/MS.

[Uwe Neue]

here is a very simple version: coelution of many analytes reduces the charge density available for your analyte, and thus the number of ionized analyte molecules.

[sam.pedraglio]

Uwe's explanation is a good starting point for the ion suppression.
The case of the matrix effect is more complicated because beside to ion suppression effect you could find also a positive matrix effect: your analyte has a higher response than from a solution in pure solvent.

[smkh]

No supression occur when change internal standard..
Thanks alot for all whom help me

[Rappoldbr]

Guys, there is a mountain of literature to deal with this - Google "Matrix effects" + any of the following names - King, Bonfiglio, Kebarle, Tang, Bennett, Grant. How to measure and/or avoid matrix effects. The steps are intuitive once you've read them, and the experiments are easy to do.