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Internal standards in regulated bioanalysis: putting in place a decision-making process during method development

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Abstract

It is common practice to utilize an internal standard (IS) to minimize variance in bioanalytical assays employing liquid chromatography coupled to mass spectrometry. For assays to be deployed in regulated drug development studies, ensuring the IS will compensate for differences in recovery, liquid handling and ionization efficiency should be determined early in the method development process. In this perspective article, we outline key considerations when selecting an IS and propose experiments to perform within the method development phase to demonstrate suitability of the IS within the assay prior to validation. Finally, a series of case studies will be presented, which illustrate analytical challenges related to internal standardization that we have observed in our laboratory.

Keywords: internal standard, liquid chromatography mass spectrometry, method development, method establishment


Introduction

For bioanalytical assays employing liquid chromatography coupled to mass spectrometry (LC–MS), the use of an internal standard (IS) as a mechanism for compensating for differences in recovery, liquid handling and ionization efficiency is well established [1,2]. Selecting an appropriate IS, and using the analyte/IS peak area ratio to quantitate, should reduce all variations in measured response apart from those due to a change in analyte concentration. A good IS will also provide useful qualitative information such as confirmation of a shift in analyte peak retention time or deterioration of peak shape, while the plot of IS responses across a batch should indicate instrument issues such as system drift. Thus, identifying a good IS is pivotal to the subsequent performance of the assay.

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Determining the appropriateness of an IS for an assay requires careful preselection followed by experimental assessment during the method development phase. To obtain the best results, the IS should be added in a miscible solvent to the sample to enable equilibration and prior to any sample extraction or modification, such as hydrolysis or derivatization, to cover the largest scope for analytical error. Thus, the IS would enable the analyte/IS peak area ratio to normalize recovery and ionization events between calibrators and samples, and correct for auto-sampler injection variance.

A key characteristic of a well-developed assay is the ability to differentiate between an expected and unexpected IS response with an unexpected result providing some basis for sample reanalysis [3]; however, due to the compensating nature of an IS, a certain level of variability in measurement is expected. While there are no set criteria for IS precision within a batch, it is generally expected that a visual assessment of a plot of IS peak areas would be sufficient to identify an analytical error, such as a miss or double spike of IS, or a systematic variation such as differences seen within a subject profile or at the same time point in many profiles [4]. Thus, an assay that displays an inherently large degree of variability in the IS response would cast doubt on the trueness of the sample measurements. Subsequently, there is value in developing methods with minimal IS peak area imprecision, regardless of the correcting power of the analyte/IS peak area ratio, as it enables outlier detection.

Acquiring the ‘ideal’ IS and developing a robust and precise method comes at a cost, both financial and in analyst time, which will need to be weighed up against the stage of drug development and the intended purpose for information generated with the assay. In any case, the time allocated to method development is limited and so the developer needs to determine the suitability of the assay within the fewest experiments possible before moving on to validation.

Click here to read the article in Bioanalysis

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Source: Wright MJ, Wheller R, Green R et al. Internal standards in regulated bioanalysis: putting in place a decision-making process during method development. Bioanalysis 11(18), 1701–1713 (2019)

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