No, we do not rely on a few MS signals alone for total abundance or 'concentration'. So called "abundance" is related to how well the various samples ionized under the specific HPLC method AND the MS parameters you selected (VERY SUBJECTIVE) relative to the baseline. Change the settings and you get different results. Relative abundance values for different spectra within the same chromatographic peak are just that, relative, and not a true measure of amounts (just the recorded values at those settings). You only sampled 3 pts within the one peak. Some samples exist in high concentrations, but ionize poorly. Some in low concentration, but ionize strongly. Some compounds exist, but are never seen. Hundreds of variation in signal detection lie between as this is a complex topic, esp for a web forum. 'Areas' or 'ion abundance' counts only have meaning when properly developed methods are used with formal standards. After that, complete calibration tables are created for quantitation (best done using a non-MS detector as MS is poor at quant). Isotopically labeled standards are best, but true "standards" in some form would be needed.

Mr. Krettler,

The quantification of mass spectrometric variable 'intensity' exactly (or absolutely) reflects the analyte concentration in solution. The currently, used concept of the so-called relative abundance or the so-called total intensity values (ITOT) of an MS peak over the whole time of the measurements does not express exactly the mass spectrometric ionization phenomena, which are random as character and are expressed via teh so-called 'fluctuations'' of the measurable variables (m/z- and intensity-values.) For this reason, the current quantitative methods accounting for the ITOT-values yield to only 98 % reliability of the data.

Precise, accurate, selective and sensitive quantification my means of mass spectrometry, currently is possible only by means of our innovative stochastic dynamic theory and model formulas (mine and my co-author's ones) which quantify the MS 'intensity' values precisely per span of scan time, instead of over the whole time of the measurements.

Our theory and formulas have been tested studying analytes in mixture at pg.(mL)-1 and ng.(L)-1 concentration levels, showing linear correlation coefficients between our theory and the experiment within /r/ = 0.99997 - 1. Our formulas and method have been validated by chromatographic experiment, as well.

Please, consider in this context references [1,2].

[1] Steroids, 164 (2020) 108750

Stochastic dynamic mass spectrometric quantification of steroids in mixture — Part II; Bojidarka Ivanova, Michael Spiteller

[2] B.Ivanova, M. Spiteller, Stochastic dynamic mass spectrometric approach to quantify reserpine in solution, (2020) in press.

I should explicitly underline, that the references [1,2] use broadly available database of experimental mass spectra, in order to, proof of our theory and innovative formulas, thus allowing their verification and proof of a broad audience, even researchers which are not experimentalist.

The absolute reliability of our theory, showing /r/ = 1 has been proven empirically examining six replicates of carbohydrate mixtures within the framework of our own experiment. Please, consider work [3].

[3] Bioorganic Chemistry, 93 (2019) 103308

A mass spectrometric stochastic dynamic diffusion approach to selective quantitative and 3D structural analyses of native cyclodextrins by electrospray ionization and atmospheric pressure chemical ionization methods

Bojidarka Ivanova, Michael Spiteller

Mr. Letter,

I am allowing myself to comment on your posting, because of it contains a misunderstanding of the physicochemistry of the mass spectrometric (MS) phenomena or the quantitative concepts of treatment of MS experimental variables or both of these, which can be found even in publications devoted to quantitative analysis by mass spectrometry.

Perhaps, the major reason for this lack of (proper) understanding is associated with the fact, that the mass spectrometry is already routinely implemented into mainly applied-scientific research areas, which use already approved ISOs and protocols.

My comment on, is associated with the following part of your text:

''No, we do not rely on MS signal alone for concentration. So called "abundance" is related to how well the various samples ionized under the specific...''

First, the term ''low abundance'' of analyte MS peak means, in fact, low intensity value.

Second, during a MS experiment, all analyte in the solution is consumed in the course of the experiment. If an analyte shows low abundance of its MS peaks, then this just means that there are observed random MS peaks of the analyte with low abundances per any of the spans of scan times over the whole time of the measurements.

Finally, if there are used classical quantitative approaches to treate the MS variable 'intensity' by means of ITOT-values, then all these sets of random MS peaks and the fluctuations of the measurable variable 'intensity' are not accounted precisely, namely, due to their low values.

However, the fact that ITOT-values do not account precisely, accurately and selectively the low abundance peaks of analyte ions, does not means that the MS 'intensity'-values do not reflect exactly the analyte concentration of the solution.

In order to clarify, I hope more illustratively, my comment on your posting, please consider the our supporting information section to work [1] shown in my posting to Mr. Krettler, stated above. In particular, look at Figure A8 showing the abundance of MS ions per scan time. These are scan times 161 and 179 of the whole time of the measurement.

Let us concentrate on peak at m/z 121 which is observed as a very low abundance ion only at scan time 179. If the quantification of the analyte is carried out only by mean the ITOT-value, then this peak should not be accounted for. By contrast, as can be seen our innovative theory and formulas account namely for any analyte MS ion per concrete span of the scan time, which, namely, makes our method highly selective. Therefore, our quantitative concept, is not only most accurate, precise and sensitive one, currently available, but also it is most selective approach to quantify the MS variable 'intensity.'

There are published only the data on the MS peaks at m/z 177, 211, 227, 267 and 285, because of the quantification of the data of five peaks is statistically significant and enough to show the exact reliability of our theory.

If you have a particular interest in detail on mass spectrometric phenomena and the developments of exact quantitative approaches to their analysis, then you could use namely the shown database in order to confirm or theory, in fact. As can be seen the software used to our work is free of charge. So, that.....

I teach MS operation and use professionally so have an in-depth understanding of the technique and its applications. Your response is not applicable to the question asked by Mr. Krettler. His question: " Is the chromatographic peak a valid measure of a compounds` (relative) abundance "; was a simple one and he only needs to understand that any signal (Peak) he accounts for may or may not represent the actual amount of sample present. The peak and its area are just the instruments response (based on his settings and method) for the peak. Abundance is only for each signal measured (using whatever settings). That is it.

Mr. Letter,

What you teach does not any matter, because of, the mass spectrometric phenomena are very complex and still many fundamental questions to many of these phenomena remain far from well understand. There is important, not what you know from the textbook, but what is your own theoretical and experimental experience as well as contributions to the methodological developments of the method.

To learn a textbook knowledge and to teach it is a high school task. 'Professor' at a University does not mean just 'teacher,' like the teachers at the High Schools. 'Professor' means academic personal dealing with scientific discoveries, generation of new scientific knowledge and their teaching to the students.

I am rather surprised to read such a comment on, from academic personal with affiliation 'Unites States of America,' but, of course, we are unable to expect that all academic personal, therein, shall be of the rank of professors such as R. Cooks, A. Marshall, R. Caprioli, et cetera. (Do you know them?! I do not need answer to this question.)

Despite, work [1*], herein, illustrates both experimentally and by means of chemometrics that the mass spectrometric intensity of the analyte MS ions corresponds exactly to the analyte concentration in solution, and to the chromatographic peak area. The work correlates absolute intensity of the MS peaks per different scan of the span time and the chromatographic data.

But the absolute MS intensity and the so-called relative abundance are mutually connected.

Therefore, what is most important for the scientific knowledge, the development of the science and the training of the students at the University: What there is written in the textbooks or what the experimental facts evidenced? The University provides a 'higher level scientific knowledge' and diplomas for Higher Education, not for High School.

[1*] Steroids, 164 (2020) 108750

Stochastic dynamic mass spectrometric quantification of steroids in mixture — Part II; Bojidarka Ivanova, Michael Spiteller

An additional comment on: Before at about 20 years I have a canceled invitation to research position at the University of Houston, due to personal reasons; my co-author is already emeritus professor, so that we perform, herein, only scientific discussion. Please, do not mix the teaching academic tasks with the academic tasks of scientific research.

Your implication that only those people who attain the title of "professor" at a school (outside the USA) are knowledgeable is laughable and very disrespectful to all people. Titles and school names alone mean nothing.To imply that you or anyone with the title of 'Professor' are superior because you have such a title shows immense arrogance. Respect is earned, knowledge and/or skills are acquired and wisdom, most of all, only attained from a deep understanding and practical knowledge, ... not gained from a title.

Many professional scientists such as myself with decades of practical research experience, who continue to make key research contributions (including many commercial scientific instruments) and work in this industry have no need for your condescending 'academic' views. We post here to provide practical knowledge, from real world use, so scientists can gain a more balanced view of how these tools work, and their real-world uses and limitations. Please do not waste our time with your unprofessional and poorly researched comments on RG.

BTW: You wrote: " I am rather surprised to read such a comment on, from academic personal with affiliation 'Unites States of America,' but, of course, we are unable to expect that all academic personal, therein, shall be of the rank of professors such as R. Cooks, A. Marshall, R. Caprioli, et cetera" - You imply that I, like you, work at a school. I have NO affiliation with any academic institution and no idea why you think I do. My experience and professional consulting services come from decades of industrial experience attained long after University training.