Should there be only 1 quantifier ion?

Discussions about GC-MS, LC-MS, LC-FTIR, and other "coupled" analytical techniques.

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When reading about quantitative analysis via MS I see that we need to:
1. Choose a quantifier ion
2. And check qualifier ions and their ratios: to see if it's really the analyte of interest

I was wondering - is it important to use only 1 ion (I assume it's almost always the base peak mass) for quantification? How acceptable is it to include additional isotopes and/or adducts when calculating peak area for quantification?

My understanding is the more masses we include - the more unrelated ions we risk to capture. But at the same time the smaller the peak area (because we chose only 1 ion) - the more impact different kinds of noise have, so the uncertainty is higher.
Software Engineer at https://elsci.io
you've answered your own question, and I agree with you!
But to add another consideration: if you are using qualifier ions, then sensitivity in terms of limit of detection is not generally determined by the quantifying ion(s) anyway. As soon as one of your qualifier ions becomes so weak that either it fails to get integrated, or if it is integrated, it fails to have an accurate enough area, then the analyte isn't detected, no matter how beautiful the quantifying peak. If the qualifiers fail, the peak is rejected (that's the point of qualifiers).
lmh wrote:
if you are using qualifier ions
Ah, so using qualifiers is optional? Since I've never seen this done myself, I'm trying to piecemeal different sources to get the whole picture. With your input included I'm getting this:

Quantifier ions are used to.. well, be included in the peak area and hence to calculate the concentration of our analyte. We can use 1 quantifier (BPM) and thus lower the risk of incorporating wrong signal. But if we're confident enough that there are no other substances with such masses - we can include more ions to make peak area measurements more precise. Typically you'd use only a few ions which give the strongest signal?

Qualifier ions should be used if there's a risk to find a peak which isn't actually our analyte of interest. I assume this is the case when:
1. The mixture content is unknown
2. Or when there are simply lots of analytes, especially those with large masses and multiple-charge species are expected.
3. Or we simply want to be extra careful

When deciding if it's the right peak we have 3 options:
1. Set min peak area (our LoD) in case we don't use qualifiers.
2. Look for presence of qualifier ions. If some of them give very low (or 0) signal - ignore this peak.
3. Or set even more strict criteria: enter (or calculate) expected isotopic pattern to look for. E.g. if we look for Cl we should expect 2 signals: 35m/z and 37m/z and their ratio must be around 66:33. If these ions are present but the ratio is 90:10 - ignore the peak.

Is this close to reality?
Software Engineer at https://elsci.io
I would say it is close, especially with the very last statement #3.

I have not worked with any software that uses more than one mass or MRM signal to quantify by area. Maybe there are some softwares that will sum the areas but most only use the quant signal for concentration calculation and other signals as qualifiers, looking to match to a range of ratios versus the main quantifier peak as a secondary test for peak identification.

How many qualifier signals to use would depend on the target analyte. Some like hydrocarbons have many fragments to choose from, some like PAH analytes only give one main fragment and then the rest are less than 10% of that in abundance which means you need a lot of the analyte to generate a positive response for the qualifier versus the response of the main fragment.
The past is there to guide us into the future, not to dwell in.
Got it, thanks!
Software Engineer at https://elsci.io
Hi:


One quantifier ion is typically used, with 1-3 qualifier ions used. The more ions you use per compound, the fewer compounds you can look for and still get the best sensitivity. So there is a trade off in terms of "quadrupole opportunity time." As you say, more ions leads to more probability of noise.


Depending on the sample matrix, interferences may "contaminate" your quantifier, but one of your qualifier ions could be free of chemical noise. In this case, you would use that qualifier as a quantifier. This is typically determined during method development: if you were developing a method to look for pesticides one part of your work would be to look at the pesticides in solvent, and then also look at them in sample matrix and look for matrix interference. If you're looking for pesticides in say rice, tea, strawberry, soil, etc you will inevitably find these types of matrix problems.


Next, it is not just about the ions. If you add retention time on your column as another dimension of the identification the MS becomes even more powerful. Commercial libraries such as NIST contain Retention Index information which can be translated to Retention Time depending on the column stationary phase polarity.

If you are doing an analysis for cocaine would you send someone to jail if you got a hit on the quantifier and 2 qualifier ions, but the retention time was 2.304 minutes and the expected retention time (confirmed with a standard) is 6.805 minutes?

For the most challenging problems there are advanced techniques like tandem MS/MS: imagine that both your analyte of interest AND an undesirable sample matrix component both make ion 200 at exactly 10.0 minutes. With a MS/MS "triple quad" instrument, you can pass mass 200 through quad 1, then fragment it a 2nd time, and pass the fragment ions of just 200 through a second quad. Once you figure out what ions your desired compound makes you can then filter out the noise even in this "worst case scenario" of chromatographic coelution + mass spec fragment ambiguity. For example: mass 200 from your analyte may fragment into masses 150, 100, and 50 while the mass 200 from your matrix fragments into 125, 75, and 50. Well, the "product ion" of 50 is still ambiguous but you can distinguish the analyte and matrix based on the others.


How far do you take this? Depends on the analysis: for very simple samples, you could simply integrate the TIC. But for very complex samples you need the chromatography playing its part and not just lean 100% on the power of the MS.
Thanks! :)
Software Engineer at https://elsci.io
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