Confused about LC-MS quntification?

Basic questions from students; resources for projects and reports.

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Hello, thanks for helping me. This seems to be a very simple to expert so apologies for that.

I worked before in LC-UV and GC-FID and it worked quite easy for quantitation as I just inject my three or four standards at different concentrations and then take each area againest concentration and plot external calibration curve to be used later for quantifying these three unknown compounds in my samples using UV based.

However, this is my first time I introduced to LC-MS or GC-MS ( actually I have not seen LC-MS yet but I would like to learn it).

I have read a lot in LC-MS instrumentation,
And how ions can be formed by API or EI etc but I still not sure how to figure from where I can quantify my compound? Do I use TIC ( i feel it is like the chromatogram obtained by LC-UV) but some people sayed this is not because TIC is a summed of all ions scaned and some mentioned SIM for quantitation( i feel this same as TIC but it is like deleting some peaks ) but a gaing do I just seperate my compound at TIC to look as resolved base line peaks then i just use their area to measure their concentration? What if not seperated do i need to seperate it to baseline?

In some books and papers I read they show two chromatogram one is intensity vs time and by pressing in each peak you will see spectra of intensity/ abundant vs m/z

Some people said use TIC for quantification

Some said use area of peaks from SIM only

Other i saw in papers never used this but they use m/z ratio , other used base peak , else used base peak divided by molecular ion.

Could you please illustrate how to quantify area? Unfortunately i am struggling to find any paper or book to expline this like what book do when they talk about UV based detection.

Thanks and I appreciate your valuable time.

Fraich
Use what you already know about GC-FID or LC-UV. It's just the same in LC-MS and GC-MS.

(1) Do I use the TIC or SIM?
Many MS instruments (e.g. quadrupoles) are much more efficient at looking at single ions than they are at collecting spectra.
(small print explanation: To collect a spectrum, a quadrupole, which is basically just a mass filter, has to scan across all the masses you want to see, which means it only allows each mass through for a tiny percentage of the total time. The detector won't see many ions. If it's monitoring a single ion, it can look at this all the while, and the detector gets a continuous stream of ions. Trap instruments generally don't benefit so much from collecting SIM data as their sensitivity is often limited by how many ions can be collected in the trap, and during the scanning process every ion in the trap is ejected at the correct time and measured. There is no filtering-out of 'unwanted' ions in a trap; instead they're sorted into mass order and measured.)
If you're using a quadrupole instrument of this sort, then you will get more sensitivity if you set it up to measure single ions.
If you set it up to collect full scan data, you can still integrate peaks from extracted ion chromatograms, which is similar to working with SIM data, but you won't get the benefit of increased sensitivity. You will, however, get better selectivity, because if a peak of mass 501 very nearly coelutes with a peak of mass 487, you will be able to look at a single peak of it on its own, rather than seeing a double-peak formed by the two masses overlapping in the chromatogram.
Summary: yes, wherever possible, quantify from single ions, and where appropriate, collect these as SIM data in the first place, rather than extracting them from scans.
(2) Do you need baseline resolution of peaks? Use the same judgement as you would in UV or FID data - the situation is the same.
(3) Spectra (intensity versus m/z) are generally used to identify compounds, while chromatograms (intensity versus time) are used to quantify.
(4) Yes, peak area in chromatograms is what you generally use, but you can also use peak height in the same way as it is sometimes used in UV or FID
(5) mz ratio: the ratio of intensity of two ions is often used, particularly in GC-MS, as confirmation of identity. You would often have a main ion which you use for quantification, and a second ion which is called a "qualifier". If its intensity isn't the same, relative to the main ion, in a sample and a standard, this implies that there is something odd going on in the sample, and the compound may not have been correctly identified. This is very commonly used in GC-MS where spectra are well-defined and reproducible, and contain lots of fragments, but it's less commonly used in LC-MS (certainly if you're using single quadrupole instruments) because there are less fragments and they're less reliable. You can use it if you are doing fragmentation in a triple quadrupole.
Hope this answers some of your questions!
Many Thanks lmh, for your great well explained answer.
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