Determination of Amantadine Residue in Honey by Solid-phase Extraction and High-performance Liquid Chromatography with Pre-column Derivatization and Fluorometric Detection

Amantadine (AMA) is an anti-viral drug used in apiculture to protect honeybee against the sacbrood virus (Morator aetatulae). This study described a reliable high-performance liquid chromatographic (HPLC) method for analyzing AMA in honey using a solid-phase extraction (SPE) cartridge (Plexa PCX) for purification, 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) as a pre-column derivatization agent, and fluorometric detection (lambda(ex)=470 nm, lambda(em)=530 nm). The chromatographic separation was performed on an XDB C18 column (150x4.6 mm i.d.) using 0.1% trifluoroacetic acid/acetonitrile (35 : 65, V/V) as the mobile phase at a flow rate of 1.0 mL.min(-1) with a run time of 20 min. Under these optimal conditions, a linear relationship was observed in the range of 0.025-1.0 mu g.mL(-1) with a good correlation coefficient (0.998) and low limit of detection (0.0080 mu g.g(-1)), the recoveries were all above 90%, and the intra-day and inter-day precision (RSD) ranged from 3.4%-5.1%.