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Abstract
In this study 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) is proposed as a post-column derivatization (PCD) reagent for the fluorescence detection of aliphatic primary and secondary amines after HPLC separation. Five primary (methylamine, isoamylamine, 2-phenylethylamine, putrescine, and histamine) and one secondary amine (dimethylamine) were separated isocratically on a cation-exchange column using HNO3 (5 × 10−3 mol L−1) as the mobile phase. Post-column derivatization was based on two steps: 1) the derivatization of amines with NBD-Cl in alkaline medium, and 2) the acidification of the resulted mixture in order to minimize the background signal of the reagent and improve dramatically the sensitivity and determination range. The variables of the post-column reaction (concentration of NBD-Cl, buffer concentration and pH, reaction temperature, concentration of HCl, flow rates of the reagents) were thoroughly investigated. Critical chromatographic parameters such as the concentration of HNO3, the percentage of organic solvent, and the column temperature were also examined to achieve adequate separation. An internal standard of 1,7-diaminoheptane was used. The developed post-column method provides the ability for a fully automated analysis, low detection limits (LODs 20–100 µg L−1 with S/N = 3), and it requires less sample preparation. The applicability of the proposed analytical scheme was demonstrated by the determination of histamine (HIS) in tuna fish tissues according to US Food and Drug Administration (FDA) guidelines.
Notes
a According to Sigma-Aldrich reagents' catalogue (2010).
a R is the ratio of peak areas of each amine versus the ISTD, [amine] is the mass concentration of the analyte in µg mL−1, S is the slope and b the intercept.
b Based on S/N = 3.
a Mean value of three replicates.
b Not detected.