Advancing Bioanalytical Method Validation: A Comprehensive ICH M10 Approach for Validating LC-MS/MS to Quantify Fluoxetine in Human Plasma and Its Application in Pharmacokinetic Studies

• Published in: Molecules (Basel, Switzerland) Switzerland), 2024-09, Vol.29 (19), p.4588
• Main Authors: El Orche, Aimen, Cheikh, Amine, El Khabbaz, Choukri, Bouchafra, Houda, Faouzi, My El Abbes, Cherrah, Yahya, Ansari, Siddique Akber, Alkahtani, Hamad M, Ansari, Shoeb Anwar, Bouatia, Mustapha
• Format: Article
• Language: English
• Subjects: Accuracy; bioanalysis; Calibration; Chromatography; Chromatography, Liquid - methods; Expected values; Fluoxetine; Fluoxetine - blood; Fluoxetine - pharmacokinetics; Formic acid; Humans; LC-MS-MS; Limit of Detection; Liquid Chromatography-Mass Spectrometry; Male; Metabolism; Metabolites; Methanol; Methods; Organic acids; Pharmacokinetics; pharmacokinetics application; Plasma; Proteins; Quality control; Reproducibility; Reproducibility of Results; Tandem Mass Spectrometry - methods; validation
• ISSN: 1420-3049; 1420-3049
• DOI: 10.3390/molecules29194588

A fast and sample cleanup approach for fluoxetine in human plasma was developed using protein precipitation coupled with LC-MS-MS. Samples were treated with methanol prior to LC-MS-MS analysis. Chromatographic separation was performed on a reverse phase column with an isocratic mobile phase of methanol and 10 mM ammonium formate pH acidified with formic acid (80:20, / ) at a flow rate of 0.2 mL/min. The run time was 4 min. Mass parameters were optimized to monitor transitions at / [M + H] 310 > > 148 for fluoxetine and / [M + H] 315.1 > > 153 for fluoxetine-d5 as an internal standard. The lower limit of quantification and the dynamic range were 0.25 and 0.25-50 ng/mL, respectively. Linearity was good for intra-day and inter-day validations (R = 0.999). The matrix effect was acceptable with CV% < 15 and accuracy% < 15. The hemolytic effect was negligible. Fluoxetine was stable in human plasma for 48 h at room temperature (25 °C), for 12 months frozen at -25 °C, for 48 h in an auto-sampler at 6 °C, and for three freeze/thaw cycles. The validated method was applied in a pharmacokinetic study to determine the concentration of fluoxetine in plasma samples. The study provides a fast and simple bioanalytical method for routine analysis and may be particularly useful for bioequivalence studies. The method was successfully applied to a pharmacokinetic study of fixed-dose fluoxetine in nine healthy volunteers.