Inconsistent internal standard response in LC-MS/MS bioanalysis: an evaluation of case studies

Area of Science:

• Analytical Chemistry
• Bioanalysis
• Mass Spectrometry

Background:

• Monitoring internal standard (IS) response is standard practice in bioanalysis via LC-MS/MS.
• Variations in IS response can question assay quality and necessitate root cause analysis.

Purpose of the Study:

• To investigate the impact of internal standard variability on bioanalytical assay results.
• To explore strategies for addressing and resolving IS response variations.

Main Methods:

• Case studies involving observed IS response variability.
• Re-analysis of diluted samples.
• Spiking of predose study samples.
• Evaluation of different labeled internal standards during method development.

Main Results:

• In two case studies, IS variability did not affect assay results, as confirmed by re-analysis and spiking experiments.
• A D17-labeled IS was found unsuitable during method development and was replaced.
• The root cause of IS variability was not always definitively determined.

Conclusions:

• Demonstrating assay accuracy and reliability is crucial, even when the exact cause of IS response variation is unknown.
• Assay re-development may be required to resolve persistent IS response variability issues.
• Careful selection of internal standards is important during method development.