Hi everyone,
I am currently using UHPLC-Q-Exactive-MS system for metabolomics analysis. Initially, I have normalized the extracted data (processed by Compound Discoverer software) using the internal standards (ISs) peak area but the data was not satisfactory.
Therefore, I wanted to try other normalisation techniques to compare my data. In many articles, researchers are using total ion count and total spectral area appraches for normalising their metabolomics data. However, I don't know how to evalute the total ion count or the total spectral area. Therefore, I need your kind valuable suggestion and guidace to evaluate it. I would appreciate anykind of assistance. Thank you.
Total Ion current is the sum of all counts in the MS over the period of acquisition of the scan. Since a peak elutes from the column over many scans, you have to sum the total ion current in each scan over the time of the peak elution. That being said, now there are things you need to consider:
1) is there more than one peak in the MS during the time period? If so, you may want to limit the mz range over which you sum the counts. Note, you can sometimes resolve two or more peaks for each peak in the LC by doing this.
2) Is the baseline constant in the MS from scan to scan? If the baseline is rising or falling over time, you will have to baseline all the MS spectra. There is no standard statistical method for this, and the method used may depend on the type of MS (TOF, TRAP, ORBITrap, or FTICR). FTICR and Orbitrap are pretty standard, TOF is the most difficult. The problem is noise is uni-directional in MS, not bi-directional. This is why signal averaging doesn't work well in MS.
3) The final issue involves isotope series. The lower abundance isotopes appear later in the MS scans and disappear sooner than the higher abundance isotopes. This means you get inconsistent peak areas (peaks area being total counts at an mz integrated over elution time). This issue makes it difficult to determine the true start and stop times of the eluted peak. You can deal with this via isotopic vector angle analysis.
Method Isotopic Vector Angle Analysis in Mass Spectrometry v2
Mr. Rashid,
Please, consider this work:
[1] B. Ivanova, M. Spiteller, Mass spectrometric stochastic dynamic 3D structural analysis of mixture of steroids in solution – Experimental and theoretical study, Steroids 181 (2022) 109001:
[https://www.sciencedirect.com/science/article/abs/pii/S0039128X22000393].
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