Hello, I'm approaching an untargeted UHPLC-HRMS analysis using ESI source in positive ionization. I am tracing the area intensity of about 20 molecules, of which we do not yet have sufficient structural information and which may be very different from each other. I am having difficulty choosing the internal standard to use in order to normalize the areas. In theory, would any molecule capable of responding adequately in the positive be suitable for normalization activity? Thank you so much, Francesco
Even using the same solvent and spray conditions, only order of magnitude response factor may be obtained with unknow analytes. It is strange that you don't have a minimum information on the sample like natural extract, impurities in drug, pollutants, which solvent (if extracted, give the polarity of compounds in sample) etc.
Hello Francesco
I think this is very difficult. The ionization of ESI ion sources is mostly very substance specific. This means that very different analytes with also very different degrees of ionization generate the signals. This response is of course reflected in the results. Even if only semi-quantitative work is done, the contents can be very far from reality.
I would first try to clarify as many structures as possible and then select one or more standards.
Which MS type do you work with? For the structure elucidation
an ion trap with MS^n would be very useful. But also a triple-quad system or Q-ToF- MS can give good hints about the structure by fragmentation.
It is also useful to look at the samples in their history and narrow down the possible analytes from that.
Good luck with your work and best regards
Joachim
Once you have some idea about the structures of the analytes, you may use some models to correct the ESI response. See the papers by Ivo Leito et al. , see the references in this paper:
I. Leito et al. Quantitative electrospray ionization efficiency scale: 10 years after. Rapid Commun Mass Spectrom. 2021;35:e9178.
Another useful piece of information about your analytes would also be the molecular formula. You write that you used a HRMS, I think a Q-ToF or Orbitrap system, so you can calculate a molecular formula for the analytes. With this you come a lot closer to the structure. In conjunction with the fragment spectra, possible candidates can be narrowed down.
Noel W Davies Thank you for your answer. Yes, you're right, it can't be defined as a semi-quantitative, actually the goal is just to normalize the intensity of the areas between runs
Joachim Horst Jean-François Gal Thank you all so much for your replies and for your kindness. I apologize for editing just now. I erroneously used the term "semi-quantitative", I am aware that in that case one or more standards are needed that have a certain structural similarity with the molecules I want to quantify. Actually, at this stage of the study, it would be useful for me to just normalize the signal of the areas between one run and another, so that I could adequately correlate the intensity of the areas between one sample and another. The choice of the internal standard is only relative to this type of activity
If you maintain strictly constant ESI(+) conditions, any (weak?) base M could be adequate to produce MH+. I fear anyway that some uneven ion suppression may occur with some molecules M (if they are too basic?). I have no experience in this regard. You may try, on a given sample, small additions of standard at different concentrations to observe the relative signals intensities.
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