Dear Nasim,

have you run replicates of that sample? Is that altered Q/I ratio reproducible between replicates? Also, is the retention time of that peak the expected one (same as in your standard solutions)?

Since you have excluded the matrix effect on the Q/I ratio, what comes to my mind is that possibly you have some kind of isomer (or even a similar compound with a slightly different mass) in your sample.

If you are using low-resolution MS such as triple quad, it might be possible that you see similar compounds all in place of your analyte of interest. Isomers of your analyte would have the same mass as your analyte, but different fragmentation patterns (same fragments, but with different abundancies).

However, I do not know what your analyte of interest is, so I do not know if its isomers could even be expected in your sample.

Based on your LC-MS/MS analysis, a good peak for the quantifier ion suggests the presence of the analyte, but a poor intensity for the qualifier ion leads to an outlier ion ratio. This could indicate matrix effects, contamination, or method issues. To address this, optimize your method, mitigate matrix effects, ensure clean sample handling, and consider using internal standards.

Zvonimir Mlinarić

No I did not run replicated of those samples, I will run them.

Retention time is the same, I mean quantifier and qualifier ions of analyte have the same retention time as standards.

Yes our instrument is QTRAP 6500+ SCIEX. I agree with your idea about isomer or any other similar compounds. I will run the same replicates with standards again, and check them.

Thank you so much for your help.

Dr Balamurugan Krishnamoorthy

Thank you so much for your help.

We have been using this method with the same instrument for 8 years. I will consider other ways to decrease matrix effect, and your other suggestion.

Nasim Alijanimamaghani

Yes, I would suggest that you first run replicates of that sample (if you have enough of the sample) just to see if this altered Q/I ratio is similar between the replicates.

If it is comparable between replicates, then I would bet on the similar compound (or isomer) in your investigated sample. Most likely it is not a constitutional isomer since it would have slightly different retention time in most cases.

Perhaps stereoisomers of your compound have the same environmental, biological, or whatever effect you are trying to demonstrate. In this case, you can label your sample as contaminated even if a different enantiomer or diastereomer is present.

Good luck!

Zvonimir Mlinarić

Dear Zvonimir,

I appreciate your help. Thank you so much. We work on mycotoxins. Actually they are toxic secondary metabolites from Fusarium species.

Nasim Alijanimamaghani

Dear Nasim,

I think that it is most likely this "isomer thing" then.

For example, the trans isomer of zearalenone which is naturally occurring can be isomerized into cis isomer under UV (which is basically anywhere not protected from sunlight).

Also, trichothecenes are a large group of similar compounds. Types of trichothecenes are chemically different, but compounds from the same type could differ only in the position of a substituent. You probably know this better than me.

These compounds would then have the same mass, and perhaps even the same fragmentation pattern, but with different abundancies. I am not saying this is what happens, but just that this could happen. It depends on the compound of course.

However, if the isomer(s) of your mycotoxin of interest were shown to have the same contaminating effect, and if you do not have to quantify the toxicity/contamination, but just say if there is contamination, then, in my opinion, you can label the sample as contaminated, even if Q/I ratio is altered.

Dear Nasim,

We have examined Fusarium toxins in cereal products in our laboratory as part of a project.

I can confirm Zvonimir Mlinarić 's statement that these toxins have structural isomers that do not or hardly differ in their retention times, masses and fragments. However, we did not focus on metabolites but only on the toxins. As MS we used the Thermo ID-X Orbitrap, so that we could calculate a sum formula for the LC-peaks with the help of the accurate mass, which then coincided with the sum formula of the toxins. The MS/MS spectra could also be found in Thermo Fisher's MZCloud database and thus the toxins could be clearly identified. This was the aim of the students' work. If you have the possibility to analyze your samples with HRMS, I would recommend it. I agree with Zvonimir Mlinarić that a shift in ionic ratios can be explained either by matrix effects or by the presence of various structural isomers. I consider the latter to be the probable cause.

Good luck with your work and best regards

Joachim

Joachim Horst

Dear Joachim,

Thanks a lot for your reply.

I agree with you that this issue is most likely because of the various structural isomers.

Since we already prepared standards is blank matrix, and we got good responses, and ion ratios for them, so we already eliminated matrix effect.

I appreciate your help.

Best wishes,

Nasim

ion ratio is a parameter used for qualitative and quantitative analysis. It refers to the ratio of the intensities of two selected ions of a target compound.

Dear Nasim,

Poor qualifier ratios could indicate either that the peak in the sample is not the compound of interest or that there is an interference with the qualifier or quantifier. You can also see poor qualifier ratio if there is saturation of the detector with high concentration. Do you just have one qualifier? Try alternative qualifiers if possible.

Hi Patrick Mccabe

For some of them, I have two qualifier ions, so yes I can try alternative qualifiers.

Another problem which I have is noise level, for LOD and LOQ:

For some of the analytes the average noise and standard deviation in the left and right side of the peak is high, so I am getting high values for LOD and LOQ (some times 120 ppb!). I am sure that we are doing everything correctly. And our instrument is also working perfectly.

The only way which comes to my mind is to filter blank matrix (clean corn) by syringe filter, after solving standards in blank matrix, keep them in fridge over night to remove any sediments, and then before transfering them to the vials filter them again by syringe filters.

Do you have any comments about this?

I appreciate your help,

Nasim