Sensitive Determination of the Binding of Antidepressants to Synthetic Melanin by Liquid Chromatography After Pre‐column Derivatization with Dansyl Chloride

Abstract

Desipramine (DES), nortriptyline (NOR), amoxapine (AMO), and maprotiline (MAP) are clinically used as antidepressants that contain a secondary amino group and are commercially available. We investigated high performance liquid chromatographic (HPLC) analysis of DES, NOR, AMO, and MAP by derivatization with dansyl chloride (Dns‐Cl) in phosphate‐buffered saline (PBS). These samples were immediately mixed with Dns‐Cl at 50°C for 30 min and injected into HPLC (excitation and emission wavelength: 370 and 506 nm, respectively). Retention times of DES, NOR, AMO, and MAP derivatives were 14.2, 15.5, 14.2, and 14.2 min, respectively. Linearity was displayed for DES, NOR, AMO, and MAP concentrations ranging from 10 to 2000 nM (r 2 = 0.9977, 0.9992, 0.9992, and 0.9994, respectively. The lower limits of detection of DES, NOR, AMO, and MAP were 5, 5, 7, and 5 nM, respectively. The coefficients of variation for their intra‐ and inter‐day assays were less than 3.6–4.4% and 3.9–4.9%, respectively. The recovery of DES, NOR, AMO, and MAP was good. DES, AMO, and MAP were not found to interfere with the peak of the NOR derivative. The binding of antidepressants to synthetic melanin was measured by determining the unbound concentration ratio to total concentration of DES, NOR, AMO, and MAP. NOR showed the strongest binding to melanin of the four antidepressants. Our results indicated that the HPLC assay of DES, AMO, MAP, and NOR by derivatization with Dns‐Cl is simple, sensitive, and reproducible in PBS. In addition our assay system is applied for the binding studies of antidepressants to melanin.