For doing LCMS analysis of metabolites from crude samples of seeds, what is the procedure for fixing internal standards? Do we do multiple screenings until we get a detectable concentration peak of the standard and then use that for further analysis?
I think we have to do multiple screenings until we get a detectable concentration peak of the standard.
Hi, I am not sure what you mean by "fixing" the concentration of an internal standard (IS). Do you want to determine the right concentration of the IS? If so, the IS should have a concentration resulting in reproducible peakareas (even after the loss oduring extraction). Therefore, I would not use concentrations which are to close to the LOD. If you are not sure which concentration is right you should test different ones. But do not use the low concentrations which result in just detectable peaks. These are normally not reproducible, especially if you consider the loss of IS during extraction. Additionally, keep in mind that your IS should be very similar (not identical) to your targets. If you look for several different substances in your samples it can make sense to use different IS. Also isotope labelled IS can make sense if have targets of special interest.
Mrs/Miss Namitha,
There is not needed to employ in internal standard in order to achieve absolute (/r/=1) quantification by means of LC-MS.
Please, consider the following most recent work [1], which has been carried out according to our own-authored (mine and my co-author's innovation according to the authorship shown below) innovative stochastic dynamic theory and model equations. Since, work [1] is submitted, please see its abstract (below) and the calibration equations (attachment, figure.)
You could pay attention to works [2,3], which use the same model equation. There is obtained, again, absolute quantification in chemometric terms, however, in mixture of standards, where there are not accounted for complex sample matrix effect, like work [1]. Despite, there is carried our correlative analysis between mass spectrometric and chromatographic data. There is /r/ = 0.99999.
[1] B. Ivanova, M. Spiteller, Stochastic dynamic electrospray ionization mass spectrometric quantitative analysis of metronidazole in human urine, (2022) submitted.
Abstract: The study reports quantitative analysis of metronidazole in clinical human urine employing our most recently developed stochastic dynamic theory and model equation D’’SD=2.6388.10-17.(–2) without presence of internal standard and within the framework of direct mass spectrometric assay. There are used ultra-high accuracy nano-electrospray ionization mass spectrometry and chemometrics. The linear calibration D”SD=f(conc.) equations are obtained within concentration range 2.5 to 25000 ng.(mL)-1 of spiked urine samples, examining a set of analyte molecular and fragment ions at m/z 171.0998, 172.0718, 172.04081, 213.1463, 181.0722, and 151.1114 , respectively. Since, there is obtained exact coefficient of linear correlation between theory and experiment looking at data on functional relation between D”SD parameters and analyte concentration in solution of ion at m/z 172.04081 (|r|=1), with this paper we attempt to illustrate that with employment of shown above formula, which quantify exactly the temporal behavior of measurable mass spectrometric variable ‘intensity’ (I) per short spans of scan time of an experiment, many problems are dealt with, that is called ‘sample matrix effect.’
[2] Analytical Chemistry Letters, 10 (2020) 703-721; Stochastic Dynamic Mass Spectrometric Approach to Quantify Reserpine in Solution; Bojidarka Ivanova, Michael Spiteller.
[3] Steroids, 164 (2020) 108750 Stochastic dynamic mass spectrometric quantification of steroids in mixture — Part II; Bojidarka Ivanova, Michael Spiteller.
Please, cosider, also the following attachments, associated with reference [1], shown in my preceding posting:
(i) MTZ-urine-equ.tiff: It contains our innovative basic equations (1) and (2);
equation (3) is according to Arrhenius's theory;
and
(ii) MTZ-urine-dqc.tiff: It contains diffusion coefficients of MTZ in urine according to reference [1] and equations (2) and (3). As can be seen, there is excellent correlation between our theory and Arrenius's one.
Once, when there are assigned MS ions to corresponding 3D molecular structures, there is not needed employment in internal standard.
There is, in actual fact, excellent-to-exact liner correlation between two completely independent theories: Our stochastic dynamic formulas, mentioned above, and the Arrhenius's one.
Thus, 172.072 belongs to N3+-cation of MTZ, while MS peak at m/z 172.041 is assigned to N+O-cation. MS peak at m/z 171 belongs to cation-radical of MTZ.
The shown equations describe completely and absolutely mass spectrometric phenomena toward (a) quantitative and (b) 3D structural analyses of analytes.
In other words, even in complex matrix like urine, there is completely able not only to determine analyte quantitatively, but also to determine its 3D molecular structure; furthermore, exactly in chemometric terms.
Dear Bojidarka B. Ivanova , thank you for sharing this innovative research. I will see if I can incorporate this methodology in my research. Since I am also planning a metabolite profiling study of crude extracts of seed embryo, as Axel Martin Orban had suggested, using just one standard won't be wise.
Thanks again, have a good day.
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