Optimization and Validation of Analytical Method for Determination of Histamine in Fish using HPLC-FLD

Article history: Received 01 July 2015 Accepted 29 July 2015 In the present study, simple and reliable method of histamine analysis was developed, optimized and validated to meet the requirements of fish safety. Histamine in fish samples was extracted with a methanol–phosphate buffer including 2 % TCA solution (50:50, v/v) followed by defatting with hexane after centrifugation at 13,000 rpm. Histamine was separated, identified by reversed phase high performance liquid chromatography (HPLC) with gradient mobile phase (methanol–phosphate buffer at pH 3.5) and quantified by fluorescence detector at excitation: 345 nm, emission: 445 nm after on-line derivatization with O-phathalaldehyde in a pre-column derivatization step using automatic injection program. The method was validated with average recovery 96% and coefficient of variation (CV %) less than 8 %. The uncertainty was estimated and was set to ±26.4 %. The performance of the proposed method was checked with a sample from the Food Analysis Performance Assessment Scheme (FAPAS) as an external quality control; the z-score was found within acceptable range │Z│< 2. Thirty four fish samples were subjected to histamine assessment using the validated method. Only one sample was contaminated with histamine and exceeding the permissible limit set by Codex Alimentarious.