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- Posts: 33
- Joined: Thu Oct 04, 2007 7:11 am
I have been thinking about the philosophy behind quantitative analysis. A typical way to perform quantitation is to make e.g. 7 dilutions of the compound of interest and include a constant amount of internal standard to those dilutions. After that, we perform the LC-MS(/MS) runs and create the calibration curve, where the y-axis is A(compound of interest)/A(internal standard), and x-axis is concentration. Here I call this type of calibration curve RRF calibration curve.
This has led me to think about the dynamic range of the internal standard itself. I assume that the most important thing is that the concentration of the internal standard is within its own dynamic range. This is because the response changes from run to run e.g. because of the sample preparation (we add the is to the samples before SPE purification), and the area of the internal standard peak naturally needs to "react" to these changes.
When planning to use a certain compound as an internal standard, I suppose I need to determine its own dynamic range by determining the minimum and maximum areas that give linear detector response. If the area of the IS peak is not within this area range, I know I am outside the dynamic range for the IS compound. Am I right in this?
I have also a philosophical question concerning the RF-type (not RRF-type!) calibration curve of the internal standard compared to the calibration curve of the compound of interest (where the y-axis is A(internal standard) or A(compound of interest), respectively, and x-axis is the concentration of the internal standard or compound of interest, respectively). Do these compounds (internal standard and the compound of interest) need to be so alike that they have the same theoretical y-axis intercept? Or does the intercept of both compounds need to be close to zero? My mathematical skills are not good enough to solve this
.
I ask this to in order to know when I (or QuanLynx) can perform quantitation by using A(compound of interest)/A(IS) ratios (RRF-type calibration curves) so that I get therotetically accurate results.
Thank you very much! I hope somebody has time to try to figure out what I mean.
This has led me to think about the dynamic range of the internal standard itself. I assume that the most important thing is that the concentration of the internal standard is within its own dynamic range. This is because the response changes from run to run e.g. because of the sample preparation (we add the is to the samples before SPE purification), and the area of the internal standard peak naturally needs to "react" to these changes.
When planning to use a certain compound as an internal standard, I suppose I need to determine its own dynamic range by determining the minimum and maximum areas that give linear detector response. If the area of the IS peak is not within this area range, I know I am outside the dynamic range for the IS compound. Am I right in this?
I have also a philosophical question concerning the RF-type (not RRF-type!) calibration curve of the internal standard compared to the calibration curve of the compound of interest (where the y-axis is A(internal standard) or A(compound of interest), respectively, and x-axis is the concentration of the internal standard or compound of interest, respectively). Do these compounds (internal standard and the compound of interest) need to be so alike that they have the same theoretical y-axis intercept? Or does the intercept of both compounds need to be close to zero? My mathematical skills are not good enough to solve this
.I ask this to in order to know when I (or QuanLynx) can perform quantitation by using A(compound of interest)/A(IS) ratios (RRF-type calibration curves) so that I get therotetically accurate results.
Thank you very much! I hope somebody has time to try to figure out what I mean.
