In my current bioanalytical method development project, I achieved optimal separation between my analyte (A) and internal standard (IS) using HPLC with fluorescence detection. However, the excitation/emission wavelengths I used for A and IS were different (272/298 nm and 360/446 nm, respectively), making use of the time program of my HPLC. Peak symmetry and area were excellent. I would like to ask if this techniques is generally acceptable by analytical chemists and/or chromatographers, before I proceed to method validation. Thank you very much!
Your ISTD should be similar in properties to your actual samples. The method used to analyze them must be the same. If you change the detector settings, then the method is no longer the same as detector settings are part of the method of analysis. *The emission and exc wavelengths used should be the same for all validated materials used in the same sample set. If they are different, then you are not comparing data from the same sets so the method would be invalid.
Note: This is a very different question than using different wavelengths for different samples (stds) in one method which is acceptable as long as each sample set (stds) are fully calibrated using their own calibration table for the respective wavelength. There are many practical example methods where different compound have different abs maxs so are calibrated in separate tables, for each std at each wavelength. Samples and relevant Stds In this case are constructed for multiple std types, at multiple levels, using different wavelengths. However, you can not calibrate using one wavelength for your stds (or set of values for FLD), then use a different wavelength for the actual sample(s). **Doing so does not follow the scientific method or good chromatography practices.
Hi William, thank you for your answer. But let me clarify that what I am describing is something like an on-line wavelength switching. The HPLC is programmed that, at a certain time during the analysis, the ex/em wavelength changes for optimal IS detection. In probably a similar fashion, LCMS uses different m/z settings for the analyte and IS. All sample sets (stds and unknowns) will use the same method, including detector program setting.
I don't see that you have much choice. Subtle changes in structure markedly affect fluorscence which is why being able switch ex/em is so important when measuring a compound and its metabolites. Do the characteristics of the IS fit all the other criteria required? Can you perform the assay without an IS? Calculate all the parameters with and without the IS data.
Hi, I agree with William. As you use 272/298 nm and 360/446 nm, respectively if something happens for the 272/298 nm values you will not see it in the IS. When you work with a FLD you must use an IS that respond at the same wavelengths 272/298 in your case. Search for a similar molecule but that is not presence in your samples. The use of a IS in not only for the chromatographic part it is also for the extraction part.
The decay of signal it not uniform in wavelengths that´s why when the FLD is verificate it is done at several wavelengths. That happend with the "old" lamps but also with the LEDs
Hi Robin and Eleutrio, thank you for your answers. I can't do without the IS, as it compensates for any imprecision that would occur in sample preparation/extraction. Also, this IS's recovery is similar to that of my analyte (about 100%). If I am able to successfully validate the method (FDA/EMEA) using these settings, would this nullify signal decay and other IS-related issues?
Dear Abel, there are different minimum requirements for validation a method depending in the area (doping, drugs of abuse, contaminants in water, contaminants in food, etc.) use the specific guide/standard for your bioassays. That will be enough.
I see no problem in using separate detection conditions for the analyte and IS. As Abel mentioned the situation is similar to mass spectrometry when we use different m/z for analyte and IS. It is common that even ionization parameters are different (fragmentor, CE, etc.) for analyte and IS.
Both sides address reasonable points here. With such different WL settings, there is a risk to miss things that affect one WL setting more than the other. However, this is not different to different m/z in MS. Usually, you dont have a problem, as long as the different signals dont drift away from each other within one measuring batch. You can (1) try to find another IS with WL closer to your analyte to minimize this risk and (2) re-inject a calibrator/control sample at the end of your measuring batch to check for any kind of drift.
Furthermore, to not rely on individual scientists' opinions, refer to appropriate guidelines and maybe consult your local authority if you have to register your method.
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