Dear to whom it may concern,
I have just read the application of the relative response factor based on internal standard in analytical chemistry. I am much interested in this factoc. However, I am quite confused about the application of this factor in bioanalysis and I have some questions as follows:
1. First one is whether we do not need to determine the linear range of concentrations of each analyte if we use this factor. Regarding this aspect, in my opinion, the linear range of concentrations of each analyte must be determined no matter what method is used and this factor is only applicable when the unknown concentration of each analyte is within this range.
2. If my opinion is right, that means we will have each calibration curve for each analyte. And if the intercept (b) of each calibration curve is not negligible, whether this factor remains applicable or not because this factor does not consider the intercept. In other hand, this factor cannot reflect the actual calibration curve.
3. In terms of the accuracy of data, between the two approaches of the calibration curve and this factor, which do you think will show more accurate data than the other? In my opinion, my answer is using the calibration curve.
The above questions are my headache and I hope that people, who are interested in this factor and experts in this field, please help me clear these questions.
Thank you so much and look forward to your kind reply.
Dear Quynh Khoa Pham
many thanks for this excellent question.
First, and this is my point of view, I consider the use of relative response factors a "last ditch resort," if
- the standards are too difficult to prepare
- the standards are too expensive
- the standards are not long term stable
- external calibration takes too much labor time, and the short cut of using relative response factors provides significant time and money savings.
-...
In other words, I prefer the "good old external standard calibration." I kind of like the internal standard calibration to compensate for drifts, and effects, e.g., created by sample preparation.
However, those are no answers to your questions.
Here we go:
Ad 1) relative response factors can only be applied if the calibration curves of both components are "identical." (define identical!) Ideally no intercept with the ordinate, and preferably linear. I have seen scenarios, where relative response factors were used for a limited concentration range, mostly because either of both components started to behave differently than the other outside of this range.
Ad 2) as above: the calibration curves for both components need to behave similarly for the concentration range investigated. This prerequisite - from my point of view - would oppose to typical intercepts, e.g., created by contamination or wrong integration. (define acceptable intercept!)
ad 3) external calibration first, then external calibration, closely followed by external calibration. ;-) As stated above, from my point of view, this should be last resort, and - the several cases I saw - people were okay with slightly inferior calibration and quantification performance. However, they benefited from the time and money savings. Something, we as analytical scientists, also have to keep in mind.
In all fairness, I saw examples for the application of relative response factors, where the analytical scientists put in a commendable amount of work and diligence, and - wow - those results turned out mind-blowing.
So, as usual, this is not a black-or-white answer; it is more in the grey-zone of things.
I hope this helped!
Good luck with your research!
Cheers,
Detlef.
Dear Quynh Khoa Pham
many thanks for this excellent question.
First, and this is my point of view, I consider the use of relative response factors a "last ditch resort," if
- the standards are too difficult to prepare
- the standards are too expensive
- the standards are not long term stable
- external calibration takes too much labor time, and the short cut of using relative response factors provides significant time and money savings.
-...
In other words, I prefer the "good old external standard calibration." I kind of like the internal standard calibration to compensate for drifts, and effects, e.g., created by sample preparation.
However, those are no answers to your questions.
Here we go:
Ad 1) relative response factors can only be applied if the calibration curves of both components are "identical." (define identical!) Ideally no intercept with the ordinate, and preferably linear. I have seen scenarios, where relative response factors were used for a limited concentration range, mostly because either of both components started to behave differently than the other outside of this range.
Ad 2) as above: the calibration curves for both components need to behave similarly for the concentration range investigated. This prerequisite - from my point of view - would oppose to typical intercepts, e.g., created by contamination or wrong integration. (define acceptable intercept!)
ad 3) external calibration first, then external calibration, closely followed by external calibration. ;-) As stated above, from my point of view, this should be last resort, and - the several cases I saw - people were okay with slightly inferior calibration and quantification performance. However, they benefited from the time and money savings. Something, we as analytical scientists, also have to keep in mind.
In all fairness, I saw examples for the application of relative response factors, where the analytical scientists put in a commendable amount of work and diligence, and - wow - those results turned out mind-blowing.
So, as usual, this is not a black-or-white answer; it is more in the grey-zone of things.
I hope this helped!
Good luck with your research!
Cheers,
Detlef.
1. You still should determine the linear range by changing the concentrations of the analyte while keep the concentration of your internal standard (IS). Derive RRF for each calibration point and calculate the average RRF. If the relative standard deviation of your achieved RRFs < 20% then use can use that average RRF over the range of analyte concentration you have investigated.
2. The intercept is not negligible usually happens when your blank is not free of the analyte signal, either by contamination or interferences. .If you blank corrected your RRF. If you want, you can also plot a calibration curve using concentration of the analyte on x versus the response ratio of analyte/IS on y. By doing this, you can assess the dynamic range of your calibration curve, and to see if it is applicable to your analysis. Personally I prefer to use stable isotope dilution calibration curve.
3. Usually RRF approach is used to simplify calculation and also practical stuffs. For example, if your analyte and internal standards are persistent, your instrument is stable, using RRF can save some times to prepare and run a new cal curve for each batch. However, I like to have a calibration curve with each batch of my analysis to acquire more reliable data.
Dear Dr. Detlef Jensen ,
Thank you so much for your intensive explanation. Thanks to your explanation, my questions are answered and I also know when I can use the relative response factor. Anyway, the calibration curve should be prioritized in any cases except for the impossible cases. In the case of constructing the calibration curve, I always use the internal standard to correct some errors during analysis whenever I can.
If convenient for you, please give me some favors because I would like to make some things clear.
''Ad 1) relative response factors can only be applied if the calibration curves of both components are "identical." (define identical!) Ideally no intercept with the ordinate, and preferably linear.''
You meant that both of the calibration curves should show the same slope in the same certain range of concentrations. In the case, if the intercept of each calibration curve cannot be negligible and different from each other but the slopes of both of the calibration curves are identical in the same certain range of concentrations, may I use the relative response factor instead of the calibration curve?
Thank you so much and look forward to your kind reply.
Best regards,
Pham Quynh Khoa.
Dear Dr. Khanh Hoang Nguyen
Thank you so much for your answers. I will keep those in my mind because they are invaluable to me and I can apply those into practice.
Thank you and hope to see your suggestions more.
Best regards,
Pham Quynh Khoa.
Dear Quynh Khoa Pham
I just realize that I was imprecise in my original wording.
Let me try to rephrase:
The slopes of both calibration curves need to be proportional over a given concentration range. So that applying the "relative factor " to the "reference" slope will give you a fairly good approximation. That means, the intercepts should ideally be "zero" - but we know that this is wishful thinking - let's say that they are negligible. We should keep in mind that relative calibration is an approximation for the reasons given above.
Looking at the above statement, it becomes obvious that even a non-linear relationship could be fitted. I leave it to the reader to conclude when this is applicable.
One piece of advice: Using shortcuts is a part of successful scientific research. Having said that: Make sure that you understand the impact of the shortcut and apply due diligence and somewhat more on the validation of this approach.
Nothing more disappointing than realizing that the results, reported for long based on an short-cut-approximation, are "wronger"...
I hope, that this time I made it somewhat clearer.
And my apology for replying not as quick as I wanted... Let's say: Daily work called.
Cheers,
Detlef.
Dear Detlef Jensen,
Thank you so much for your kind reply. Thanks to your explanation, I totally understand the relative response factor and know when this factor can be applicable.
Thank you so much again,
Pham Quynh Khoa.
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