Hello Francersco,

that the signal intensity is lower in PRM mode is quite normal, because here only a very small section of the masses is detected. The advantage is the high selection of the analyte signal. With an isolation width of 1.5 u, only the analyte is used in the measurement and matrix components are masked out. The selectivity is also noticeable in the signal-to-noise ratio, which improves and does not drop so steeply as the concentrations become smaller. The advantage of a high selectivity is the (mostly) interference-free

analysis, which is more important for complex matrices.

Many greetings

Joachim

Joachim Horst, thank you so much for your reply.

In fact, all of this is clear to me, I don't understand why, despite the selectivity and the ability to mask the other analytes present in the matrix, the quality of the peak is clearly lower than that of the Full Scan analysis on the same sample. As you can see in the figure in the comparison between the 2 peaks, the number of points used to build the curve in PRM is much lower.

This is about to duty cycle/dwell time frequency of the mass analyzer...You should optimize the three parameters experimentally to get similar peak results. In your method builder carefully adjust first the expected LC peak width to get the ideal number of points for ideal peak shape. Thıs directly links to the AGC target which operates the C-trap to collect ions and to gain maximum gains. Secondly, the Microscan option is crucial. You may decrease and observe the change because this controls the number of scans in a particular spectrum...

PRM and full scan creates different modes of ion collection in C-trap and subsequently, injection to orbitrap analyzer, resulting reasonably different number of points for a certain peak...The lower number of precursor ions in PRM mode due to the following fragmentation decreases the sensitivity this also affects the accumulated number in a certain time in the C trap. Thus lower signal and bad peak shape occur in the experiment.

I hope this clarifying enough for a complex issue...

Thank you very much İsmail Emir Akyildiz , I will try to follow your instructions carefully. Yes, It's a complex issue, I've already tried to make changes to the method (but your information has better focused me). I've always kept the "PRM" as a reference for quantification, but I realize that it needs an effective setup so that it can give optimal results

Hi Francersco,

Were you able to solve the issue? I am having a simillar problem. I would really appreciate if you could share how did you solve it.

Amin Sajid,

I solved this problem, improving the quality of the peak, focusing more on the collision energies and on the management of AGC Target.

In particular, I no longer used the stepped-N(CE) method but I focused on a single normalization energy N(CE). For example, for the molecules of my interest a good N(CE) was equal to 15. Instead, with the stepped-N(CE) (e.g. 20,40,70) the high energies negatively influenced the quality of the final spectrum.

Finally, to favor the accumulation of charges while reaching the AGC Target, I recommend setting the Injection Time (maxIT) slightly below the Transient Time. eg. for 35,000 Transient time = 128ms, you can set (maxIT) = 100ms. These steps have vastly improved the quality of peaks.

I hope I was clear and helpful,

Let me know if it works for your tests too, good luck.

Francesco Spataro

Thank you for sharing! It will be very helpful.

Francesco Spataro

One more question. What software do you recommend for quantification of PRM data? Thank you for your time.

Amin Sajid I used for manual integration of peaks Chromeleon or Freestyle (both are Thermo Scientific software).