2.7. Determination of Biogenic Amine Content

Biogenic amines in samples and analytical standards were determined using high performance liquid chromatography (HPLC) and the Waters AccQ-Fluor^TM fluorescent tagging system (Milford, MA, USA), a method developed for the determination of amine compounds in foods. The Agilent model 1100/1200 HPLC system included a quaternary pump, autosampler, column heater, fluorescence detector, and Chemstation™ software. Approximately 1 mL of crap sauce was filtered through a 0.45 μM nylon syringe filter (Cole-Palmer, Vernon Hills, IL, USA). Ten μL of sample filtrates and 10 μL of standards were prepared for HPLC analysis with the AccQ·Fluor^TM kit and assayed using the HPLC column and eluents supplied with the kit. All procedures included in the kit directions insert were followed, with a slight modification of the HPLC gradient elution. Standard curves were constructed using five concentrations of histamine, agmatine, putrescine, cadaverine, and tyramine (all from Sigma-Aldrich, St. Louis, MO, USA), ranging from 0.557–0.894 mg mL⁻¹ and diluted with HPLC-grade water. Baseline separation of the target analytes was achieved and biogenic amines were identified by comparing retention times from samples with the analytical standards. Peak areas were used to calculate analyte concentrations. Data were expressed as mg 100 mL⁻¹.