Bioequivalence and Bioavailability Forum

Dear Charl,

❝ " it is not necessary to reanalyze samples analyzed before optimizing the standard curve or QC concentrations."

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❝ does that means that if I evaluated the first set of 6 batches on a linearity range narrower than it should be data will still accepted?

The discussions initiated by the FDA at Crystal City were regarding situations they met where the study was initiated with a range that proved during the study to be too wide. With a MQC at mid-range and a HQC in the upper range of concentrations you may then end-up with only one QC (LQC) in the range of concentrations measured during the study. Or the opposite: LLOQ very low compared to measured concentrations, then LQC and possibly MQC below most concentrations.
FDA's recommendation was to use the correct range right from the start and part of the audience had to remind them that the guideline applies not only to BE trials (where you may indeed know what concentrations you can expect), but also to pre-clinical and early human development, where you have no idea. The final recommendation was to adapt the range after the first few runs, but not to repeat the analysis of these first runs. Your hypothesis (range too narrow during the first runs) was not discussed. IMHO: data from the first runs that are within the calibration range remain acceptable; samples outside this range are to be reassayed after dilution (if too high) or after widening the range; widening the range requires additional validation of your method.

❝ 2- page E34 INCURRED SAMPLE RE-ANALYSIS

❝ "the study sample results obtained for establishing incurred sample reportducibility may be used for comparison purposes, and do not necessarily have to be used in calculating reported sample concentration."

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❝ what does it mean????

The FDA wants you to demonstrate the precision of your method using real-life samples, not only spiked samples. Meaning that if you want to comply with this requirement you will have to re-assay some of the samples from your study. The idea is to use the results of the first determination for your PK analysis, and have a table somewhere listing the results of whatever you will have done to demonstrate precision on incurred samples. You do not need to treat these results as per your procedure on repeat analysis to select which result should be reported.

❝ 3- page E 36 Source data documentation

❝ "Modification of calibration response (deletion of individual standard points that exceed predefined acceptance limits or alteration of the standard curve range"

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❝ can I conclude that in my final study report I can delete some unaccepted points from my linearity range, and report it as (blank), ? what MODIFICATION means to u??

As per the FDA guideline you can delete a standard from your calibration curve if the back-calculated concentration deviates from the nominal concentration from more than 15 % (20 % for LLOQ). The usual process is not to report it as (blank) but to report the back-calculated concentration, with an asterisk or any mark leading to a note identifying it as excluded. Also don't forget the end of the sentence: Modification of calibration response [...] should be documented with sufficient detail to demonstrate that the changes were justified and/or followed established procedures. Remember that the 15% / 20 % is to be calculated before the standard is excluded, not after ! Exclusion of a standard will change the calibration curve parameters and the % deviation of a standard will be modified (increased) after its exclusion.

❝ 4- page E 37 STABILITY RECOMENDATIONS

❝ "...intended (nominal) concentrations should be used..."

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❝ Why we dont report stability in absolute peak area, thus reporting in concentrations and comparing with nominal values will depend also on the sensitivity of the method and the interferance of slope variation will account then...

It depends what stability experiment you are considering... For stock solution stability, or on-injector stability, the use of peak area may be of interest. But for long-term stability of plasma samples ? If you're not using the internal standard you will run into trouble.

❝ 5- page E38 MATRIX EFFECTS FOR MS-BASED ASSAYS

❝ "presence of matrix ions and absence of matrix ions"

❝ does this means to u that comparision is done between spiked plasma samples and spiked mobile phase samples??

I can't remember matrix effect studies being discussed in detail during the meeting. There are much better and more detailed papers available on this topic...

Regards
Ohlbe