Very complex internal standard response variation in LC-MS/MS bioanalysis: root cause analysis and impact assessment

Area of Science:

• Analytical Chemistry
• Bioanalysis
• Mass Spectrometry

Background:

• Internal standards (ISs) are vital for quantitative bioanalytical methods.
• ISs correct for analytical response variations due to experimental conditions.
• Variability in IS response can indicate issues in sample handling or analysis.

Purpose of the Study:

• To investigate complex variations in internal standard (IS) responses.
• To identify analytical and matrix-related root causes of extreme IS variability.
• To propose a decision tree for diagnosing IS variability issues.

Main Methods:

• Case study analysis of complex IS response variations.
• Identification of analytical problems (e.g., injection errors, sample inhomogeneity).
• Assessment of matrix-related issues (e.g., degradation, ionization efficiency).

Main Results:

• Complex IS variability can stem from injection errors, sample inhomogeneity, degradation, and increased ionization efficiency.
• These issues significantly impact the reliability of quantitative bioanalytical methods.
• A systematic approach is needed to diagnose the source of variability.

Conclusions:

• Extreme internal standard variability in LC-MS/MS methods signals underlying analytical or matrix problems.
• A structured diagnostic approach, like the proposed decision tree, is essential.
• Addressing these root causes ensures the development of robust and reliable bioanalytical methods.