On September 4, 2019, FDA issued final guidance on the Evaluation of Internal Standard Responses during Chromatographic Bioanalysis. The guidance gives recommendations to sponsors, applicants, and contract research organizations regarding internal standard (IS) response variability in chromatographic analytical data submitted in INDs, NDAs, ANDAs, BLAs, and supplements.
The guidance begins by noting that, depending on the source, IS response variability may impact the accuracy of analyte concentration measurements. The guidance also identifies several common sources of IS response variability, such as human error in preparing the standards, instrumental issues that may occur during analysis of the standards, and matrix effects within the standards.
In a question and answer format, the guidance provides examples of when IS response variability is likely and not likely to impact the accuracy of the reported data. For example, if the range of IS response for subject samples is similar to the range of IS responses for calibration standards and quality control samples, the IS response variability is not likely to impact the accuracy of the data. If, on the other hand, the IS responses are substantially different than responses for the other samples, and the calibration standards and quality control samples do not demonstrate similar variability, the IS response variability can impact the accuracy of the data. The guidance suggests three general categories of problematic IS response variability: (1) IS responses significantly different than the responses for calibration standards and quality control samples, (2) IS responses that gradually drift, and (3) IS responses that are consistently lower or higher than the IS responses for calibration standards and quality control samples.
For each observed variance in IS responses, the guidance suggests approaches that sponsors can adopt to mitigate the variability. For category 1 variances, pre-established criteria for repeat analysis should be followed on such IS responses that significantly differed. For drifting variance, category 2, subject samples displaying the aberrant IS responses should be reanalyzed. The guidance then offers further steps that sponsors should take based on the results of the reanalysis. Finally, for category 3 where constantly lower and higher IS responses are observed, a subset of subject samples should be reanalyzed. Again, the guidance offers further steps to be taken based on the results of this reanalysis.