Chromatography Forum

I perform a validated LCMS/MS assay in rodenticide analysis using surrogate IS (dicoumarol). The test was all along ok. Recently, the calibration standards responses fluctuate: sometimes similar S0-S3 IS response but high (doubled) S4 IS, sometimes randomly high and low IS among different standard points. In one occasion, all standard points responses were similar and satisfactory.

The QCs (made from a different pool of serum) IS were significantly lower than standard IS. Likewise, patient sample IS was significantly lower than standard IS (half that of standards').

The molecular weight of the IS is 336 and should not be easy to be blown away during TurboVap evaporation. New reagents, mobile phase were used. Repeat sample preparation by different operator showed similar phenomenon.

Any idea how to troubleshoot the problem is deeply appreciated.
Thanks.

Have you guys validated the assay in terms of ion suppression/enhancement?? You could have some matrix effect going on?

The assay has been in use for some years, but the standard curve IS variation problem happened only recently.
QCs IS responses used to be similar to standards IS too.

Apart from that are you guys using the same batch of blank serum from long ago, or is that different.

I really think you guys should do a simple post column infusion of the I.S. using a sample T,try to match your baseline to the abundances of the usual spiked amount, then simultaneously inject your blank serum, ensure there is no significant supression or enhancement in the region of interest.
You talk about the IS response fluctuations, I have to ask if the analyte itself is behaving similarly?

On another note, looking at the MS itself,

For the I.S. why dont you try monitoring another product ion of the same precursor, to see if this phenomena is obtained.

It seems to me there is nothing wrong with the sample prep and LC seperation, but that you have something going on in the MS settings, probably something going on in the source and ion production.

Yes, there is a change in blank serum lot for spiking standards recently.
I am spiking the standard with another blank serum matrix.
I'll update the progress when available.
Thanks.

Best of luck my friend.

It turned out that the IS responses were constant when an activated charcoal treated blank serum matrix was used.
It seems that the commercial blank serum matrix used previously for standards somehow cause a variation in IS responses, resulting in high response in S4 (doubled) while the other standards were comparatively low.

I shall repeat once by another operator and place the standards randomly in TurboVap during evaporation to check if this is the real cause.

Thanks.