A New Precolumn Derivatization LC‐MS/MS Method for the Determination of Methoxamine Enantiomers in Rat Plasma: Application to Stereoselective Pharmacokinetic Analysis

ABSTRACT Methoxamine is a selective α‐adrenergic receptor agonist used to manage hypotension during anesthesia and prevent cardiac arrhythmias. Erythro‐methoxamine, a racemic mixture, is clinically administered in this form. Previous studies revealed that the (1 R ,2 S )‐enantiomer exhibits stronger pharmacological activity than the (1 S ,2 R )‐enantiomer, highlighting the need for stereoselective pharmacokinetic evaluation. Given the high sensitivity, excellent stereoselectivity, and resistance to racemization exhibited by ( S )‐ N ‐(4‐nitrophenoxycarbonyl) phenylalanine 2‐methoxyethyl ester [( S )‐NIFE], this study pioneered its novel application as the chiral derivatization reagent for methoxamine prior to chromatographic analysis. Based on precolumn derivatization, a sensitive, efficient, and straightforward LC‐MS/MS method was developed to determine the two enantiomeric derivatives of erythro‐methoxamine. DHBA was selected as the internal standard. After protein precipitation, the derivatives were analyzed in positive ESI‐MRM mode. The method showed good linearity over 0.5–250.0 ng·mL −1 with an LLOQ of 0.5 ng·mL −1 . Intraday and interday RSDs were below 9.7%, and recoveries exceeded 86.2%. The validated method was successfully applied to the first stereoselective pharmacokinetic study of erythro‐methoxamine in rat plasma. Significant differences in AUC, t 1/2 , Vd, and CL were observed, confirming its suitability for in vivo pharmacokinetic analysis and providing evidence for the stereoselective disposition of methoxamine enantiomers.