The release of the ICH M12 Guideline on Drug Interaction Studies has reignited discussions around assay validation requirements for in vitro assays such as plasma protein-binding studies. Even though the ICH M12 does not directly reference the ICH M10 Guideline on Bioanalytical Method Validation and Sample Analysis, its release prompted further discussions on assay validation requirements for these studies during the 17th European Bioanalysis Forum Open Symposium held in Barcelona, Spain, from 19 to 21 November 2024, where we advocated for a Context-of-Use driven approach over rigid adherence to ICH M10 standards. Context-of-Use driven validation ensures assays are tailored to the specific scientific and regulatory needs, optimizing resource allocation and innovation in drug development. This short opinion paper explores the potential and undesired implications of ICH M12 on bioanalytical validation practices, highlights the distinction between exploratory assays and assays having a clinical impact, and underscores the necessity for tailored validation strategies.

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.

AIM:To develop and validate an LC-MS/MS assay for the quantification of digoxin in human plasma. An LLOQ of 10 pg/ml using a 100 μl sample was required to support drug-drug interactions studies.RESULTS:Digoxin formed multiple precursor ions in positive and negative ESI and methods based on several of these have been reported previously. After screening viable precursor ions, we found the ammonium adduct gave the best combination of sensitivity and selectivity on our LC-MS/MS platform. Samples were extracted using a simple liquid-liquid procedure.CONCLUSION:The assay was successfully validation to current EMA guidelines. To the best of our knowledge the developed assay is the most sensitive published to date.

Aim: LC-MS/MS bottom-up quantitation of proteins has become increasingly popular with trypsin as the most commonly used protease. However, trypsin does not always yield suitable surrogate peptides. An alternative enzyme, Glu-C, was used to generate surrogate peptides for quantifying a bispecific IgG1 biotherapeutic antibody in preclinical matrices. Materials and methods: IgG1 was quantified by pellet digestion using an Acquity UPLC coupled with a Xevo TQ-S mass spectrometer. Results: Two generic LC-MS/MS methods (heavy and light chain) were developed which afforded acceptable precision and accuracy, and an lower limit of quantitation of 1 mu g/ml in three preclinical matrices. A small nonsignificant bias was observed when cynomolgus serum LC-MS/MS results were compared with electrochemiluminescent immunoassay data. Conclusion: Glu-C was successfully used as an alternative digestion enzyme for bottom-up quantitation of an IgG1 in matrices from multiple preclinical species, with good agreement with electrochemiluminescent immunoassay data.