Synthesis and Characterization of 2-(1-Hydroxyethyl)Promazine Sulfoxide-d₄ HCl as an Internal Standard for the Accurate Quantitation of 2-(1-Hydroxyethyl)Promazine Sulfoxide in Equine Urine

Acetylpromazine, 1-{10-[3-(dimethylamino)propyl]-10H-phenothiazin-2-yl}ethenone, C₁₉H₂₂N₂OS, 326.46 g·mol^-1 is a phenothiazine derivative at one time used in human medicine as an antipsychotic medication but now predominantly used in veterinary medicine as a sedative/tranquilizer and referred to as acepromazine. In performance horses its use is regulated by using a 10 ng/mL threshold for the major urinary metabolite 2-(1-hydroxyethyl) promazine-sulfoxide (HEPS) in equine urine. To enable accurate quantitation of HEPS in equine urine we have synthesized and purified hydroxyethylpromazine sulfoxide-d₄ (HEPS-d₄) to be used as a stable isotopically labeled internal standard. Although labeled HEPS is commercially available (CAS 1346605-30-8), to the best of our knowledge there is no published synthetic procedure in the scientific literature. Here we demonstrate a viable synthetic procedure consisting of four major steps: (i) freebasing the Acepromazine maleate salt, (ii) H-D exchange of Acepromazine at room temperature, (iii) reduction of the ketone with NaBD₄, and (iv) oxidation of the thioether via hydrogen peroxide and acetic acid. This deuterated internal standard will allow for precise LC/MS quantitation of HEPS at regulatory threshold concentrations, enabling accurate detection and quantitation of picogram/mL concentrations in equine urine samples, thereby supporting regulatory compliance for equine medication control programs.