Advancing Bioanalytical Method Validation: A Comprehensive ICH M10 Approach for Validating LC-MS/MS to Quantify...

Area of Science:

• Analytical Chemistry
• Pharmacokinetics
• Bioanalysis

Background:

• Accurate quantification of fluoxetine in biological matrices is crucial for pharmacokinetic and bioequivalence studies.
• Existing methods may lack the speed and simplicity required for high-throughput analysis.

Purpose of the Study:

• To develop and validate a fast and efficient bioanalytical method for fluoxetine determination in human plasma.
• To assess the method's suitability for routine clinical and research applications.

Main Methods:

• Protein precipitation using methanol followed by liquid chromatography-tandem mass spectrometry (LC-MS-MS).
• Chromatographic separation on a reverse-phase column with an optimized isocratic mobile phase.
• Mass spectrometry parameters optimized for sensitive detection of fluoxetine and its deuterated internal standard.

Main Results:

• The method demonstrated excellent linearity (R² = 0.999) and a lower limit of quantification of 0.25 ng/mL.
• Acceptable matrix effects (CV% < 15) and accuracy (accuracy% < 15) were achieved.
• Fluoxetine stability was confirmed under various storage conditions, including long-term freezing and freeze-thaw cycles.

Conclusions:

• A fast, simple, and robust LC-MS-MS method for fluoxetine quantification in human plasma was successfully developed and validated.
• The method is suitable for routine bioanalysis and particularly valuable for bioequivalence studies.
• The method was effectively applied in a pharmacokinetic study involving healthy volunteers.