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.
You have to determine the working (in your terms, linear) range of all analytes relative to internal standards. You should analyze your analyte against your internal standard at several concentrations and determine the RRF from that. If your RRF is essentially the same across the range you choose, normally defined as
Dear Mark Krause,
Thank you so much for your information.
I am now so curious about the technique of an isotope dilution. However, I do not know exactly what you meant. You meant that if I use the stable isotope internal standard of the analyte of interest, I do not need to determine the linear range of concentrations of the analyte of interest relative to this internal standard. Instead, I just use a single point to determine the RRF and then, RRF can be applicable to any concentration levels of the analyte of interest. If my guess is right, I think that it is impossible because the outside of the linear range of concentrations of the analyte of interest, its response will not be linear to its concentrations and in the case, RRF is useless. In contrary, if I misunderstood what you meant, please help me clear this issue.
Even though, the stable isotope is always considered as the internal standard of choice for any analytes of interest because it is assumed to show the same behaviors as the analyte of interest, we had better check the assumption because the internal standard is not the analyte of interest and it can show different behavior to any influence that will affect the data reliability.
Thank you so much and hope to see your kind reply.
Best regards,
Pham Quynh Khoa
Dear Pham
Check my papers - there are examples of application of deuterated IS (papers on PAHs, O-VOCs and VNCs).
Regards
GB
Like everything the answer depends. Biomolecules are expected to meet the same validation and regulatory requirements as small molecules (see the USP monographs for Insulin and Oxytocin).
HOWEVER, if you can scientifically justify differences in quantitation (linearity precision, and accuracy) you can use another system (like just using peak 12? for PCB quantitation). Remember, enzymes (a protein) require an energy source (ATP...), and chemical substrate.... So 1 molecule of an enzyme can catalyse 1000s of reactions before it is exhausted. Thus, we talk about activity and not mass!
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