Indirect enantioseparation of fluoxetine in mouse serum by derivatization with 1R‐(–)‐menthyl chloroformate followed by ultra high performance liquid chromatography and quadrupole time‐of‐flight mass spectrometry

Here we describe a simple and sensitive analytical method for the enantioselective quantification of fluoxetine in mouse serum using ultra high performance liquid chromatography with quadrupole time‐of‐flight mass spectrometry. The sample preparation method included a simple deproteinization with acetonitrile in 50 μL of serum, followed by derivatization of the extracts in 50 μL of 2 mM 1R‐(–)‐menthyl chloroformate at 45ºC for 55 min. These conditions were statistically optimized through response surface methodology using a central composite design. Under the optimized conditions, neither racemization nor kinetic resolution occurred. The derivatized diastereomers were readily resolved on a conventional sub‐2 μm C₁₈ column under a simple gradient elution of aqueous methanol containing 0.1% formic acid. The established method was validated and found to be linear, precise, and accurate over the concentration range of 5.0–1000.0 ng/mL for both R and S enantiomers (r² > 0.993). Stability tests of the prepared samples at three different concentration levels showed that the R‐ and S‐fluoxetine derivatives were relatively stable for 48 h. No significant matrix effects were observed. Last, the developed method was successfully used for enantiomeric analysis of real serum samples collected at a number of time points from mice administered with racemic fluoxetine.