Ultrasound-assisted chiral derivatization of etodolac with (1R)-(−)-menthyl chloroformate for the determination of etodolac enantiomers

This study presents the first report of an ultrasound-assisted derivatization reaction between a carboxylic acid of etodolac (ETO) and a chiral derivatization reagent, (1R)-(−)-menthyl chloroformate (R-MCF). Fifty μL of deproteinated mouse serum containing ETO enantiomers was derivatized with 125 μL of 200 mM R-MCF and 17 μL of pyridine (a catalyst), with the reaction facilitated by ultrasonic radiation for 13 min, which were the optimal conditions as determined by response surface methodology. After quenching the reaction by adding an aqueous L-proline solution, the mixture was subjected to salting-out assisted liquid–liquid extraction (SA-LLE), which provided phase separation for sample concentration as well as cleanup. The ETO diastereomers were separated on a Phenomenex Gemini C₁₈ column (150 × 4.6 mm, 5 μm) under a simple gradient elution of a mobile phase containing a mixture of methanol: acetonitrile (10:1, V/V) and 10 mM acetic acid at a flow rate of 1.0 mL min⁻¹, followed by fluorescence detection with excitation and fluorescence emission wavelengths of 235 nm and 345 nm, respectively. The developed method was validated for specificity, sensitivity, linearity, accuracy, precision, stability, and matrix effect. A good linearity in the range of 0.5–50.0 μg mL⁻¹ for each ETO enantiomer with r² > 0.998 and acceptable values for the intra-day and inter-day accuracy and precision as well as negligible matrix effects supported the suitability and reliability of the method. Finally, this method was used to analyze real samples taken from mice treated with (±)-ETO.