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
Aimen El Orche,
Amine Cheikh,
Choukri El Khabbaz,
Houda Bouchafra,
My El Abbes Faouzi,
Yahya Cherrah,
Siddique Akber Ansari,
Hamad M. Alkahtani,
Shoeb Anwar Ansari,
Mustapha Bouatia
Affiliations
Aimen El Orche
Laboratory of Drugs Sciences, Biomedical Research and Biotechnology, Faculty of Medicine and Pharmacy, Hassan II University of Casablanca, B.P. 9154, Casablanca 20250, Morocco
Amine Cheikh
Center for Bioequivalence Studies of the Sheikh Zaid Foundation, Av. Allal Al Fassi, Rabat 10000, Morocco
Choukri El Khabbaz
Center for Bioequivalence Studies of the Sheikh Zaid Foundation, Av. Allal Al Fassi, Rabat 10000, Morocco
Houda Bouchafra
Laboratory of Drugs Sciences, Biomedical Research and Biotechnology, Faculty of Medicine and Pharmacy, Hassan II University of Casablanca, B.P. 9154, Casablanca 20250, Morocco
My El Abbes Faouzi
Laboratory of Pharmacology and Toxicology, Biopharmaceutical and Toxicological Analysis Research Team, Faculty of Medicine and Pharmacy, Mohammed V University, Rabat 10000, Morocco
Yahya Cherrah
Center for Bioequivalence Studies of the Sheikh Zaid Foundation, Av. Allal Al Fassi, Rabat 10000, Morocco
Siddique Akber Ansari
Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, P.O. Box 2457, Riyadh 11451, Saudi Arabia
Hamad M. Alkahtani
Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, P.O. Box 2457, Riyadh 11451, Saudi Arabia
Shoeb Anwar Ansari
Department of Drug Science and Technology, University of Turin, 10124 Turin, Italy
Mustapha Bouatia
Laboratory of Analytical Chemistry, Team of Formulation and Quality Control of Health Products, Faculty of Medicine and Pharmacy, Mohammed V University, Rabat 10000, Morocco
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, v/v) at a flow rate of 0.2 mL/min. The run time was 4 min. Mass parameters were optimized to monitor transitions at m/z [M + H]+ 310 > > 148 for fluoxetine and m/z [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 (R2 = 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.
